Tafa4 compounds and uses thereof for treating pain

ABSTRACT

The present invention relates to a method of using a TAFA4 protein or an agonist thereof for preventing, alleviating or treating pain in a subject. In one embodiment, the invention provides a method of treating pain in a subject by administering a TAFA4 protein or an agonist thereof to the subject. The TAFA4 can have the amino acid sequence of SEQ ID NO: 1 or 2 or a sequence having at least 90% sequence identity to SEQ ID NO: 1 or 2. The TAFA4 agonist can also be a peptide comprising 10 to 60 consecutive amino acid residues of SEQ ID NO: 1 or 2. Also described herein are pharmaceutical compositions, their preparation and uses as well as methods for preventing, alleviating or treating pain using such compounds and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/787,483, filed Oct. 28, 2015, which is the U.S. national stageapplication of International Patent Application No. PCT/EP2014/059247,filed May 6, 2014.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Oct. 20, 2015 and is 8 KB. The entire contents ofthe sequence listing is incorporated herein by reference in itsentirety.

The present invention relates to novel compounds for use for preventing,alleviating or treating pain in a subject. Also described herein arepharmaceutical compositions, their preparation and uses as well asmethods for preventing, alleviating or treating pain using suchcompounds and compositions.

BACKGROUND OF THE INVENTION

Pain is an unpleasant sensory experience associated with actual orpotential tissue damage. Thus, pain is the most common symptom ofvarious injuries and diseases. There exist different classifications ofpain, for example, nociceptive pain, inflammatory pain associated withtissue damage and the infiltration of immune cells, and pathologicalpain, which is a disease state caused by damage to the nervous system(i.e., neuropathic pain) or by its abnormal function (dysfunctionalpain, as in fibromyalgia, irritable bowel syndrome, tension typeheadache, etc.). Pain is usually transitory, lasting only until thenoxious stimulus is removed or the underlying damage or pathology hashealed, but some painful conditions, such as rheumatoid arthritis,peripheral neuropathy, cancer and idiopathic pain (pain that persistsafter the trauma or pathology has healed, or that arises without anyapparent cause), may persist for years. Pain that lasts a long time iscalled chronic, and pain that resolves quickly is called acute.Traditionally, the distinction between acute and chronic pain has reliedupon an arbitrary interval of time from onset, the two most commonlyused markers being 3 months and 6 months since the onset of pain (Turk,Okifuji, Pain terms and taxonomies of pain; In: Bonica, Loeser, Chapman,Turk, Butler, Bonica's management of pain. Hagerstown: LippincottWilliams & Wilkins, 2001), though some theorists and researchers haveplaced the transition from acute to chronic pain at 12 months(Spanswick, Main, Pain management: an interdisciplinary approach.Edinburgh: Churchill Livingstone, 2000). Others apply “acute” to painthat lasts less than 30 days, “chronic” to pain of more than six months'duration, and “subacute” to pain that lasts from one to six months(Thienhaus, Cole, Classification of pain. In: Weiner, Pain management: apractical guide for clinicians. Boca Raton: CRC Press, 2002). A popularalternative definition of chronic pain, involving no arbitrarily fixeddurations, is “pain that extends beyond the expected period of healing”(Turk, Okifuji, 2001, Pain terms and taxonomies. In Loeser, Butler,Chapman, et al. Bonica's management of pain, LippincottWilliams&Wilkins. ISBN 0-683-30462-3). Chronic pain may be classified ascancer pain or benign (Thienhaus, Cole, 2002, Classification of pain. InWeiner, Pain management: A practical guide for clinicians, AmericanAcademy of Pain Management, ISBN 0-8493-0926-3).

Pain sensation is conveyed to the brain by sensory neurons which arealso called nociceptors. Nociceptors are considered polymodal since theymay respond to multiple forms of noxious or intense stimuli, such asheat, mechanical, and chemical stimuli. Sensory afferent fibers ofnociceptors are heterogeneous in many aspects. For example, sensorynerves are classified as Aα, -β, -δ and C-fibers according to theirdiameter and degree of myelination. Then, sensory inputs from theperiphery are processed and conveyed to higher brain regions by complexcircuits involving excitatory and inhibitory interneurons within thespinal cord (Basbaum et al., 2009; Todd, 2010). The balance betweenexcitation and inhibition is crucial for maintenance of normal sensoryfunction, and dysfunction of these circuits leads to the development ofpain such as inflammatory and neuropathic pain.

Treatment of pain includes the use of local anesthetics, which blockneuronal transmission and affect sensation as well as pain, andanalgesics, which relieve pain and additionally may interfere with theactivity of chemical mediators of inflammation. Acute pain is usuallymanaged with medications such as analgesics and anesthetics. Managementof chronic pain, however, is much more difficult.

The effectiveness of analgesics relies on how they are able to block thenerve messages that are sent by the pain receptors to the brain. Theyfurther have an effect on the body temperature to increase it (known asfever) or decrease it. Analgesic drugs act in various ways on theperipheral and central nervous systems; they include paracetamol(para-acetylaminophenol, also known as acetaminophen or simply APAP),the non-steroidal anti-inflammatory drugs (NSAIDs) such as thesalicylates, and opioid drugs such as morphine and opium.

The exact mechanism of action of paracetamol/acetaminophen is uncertain,but it appears to act centrally rather than peripherally (in the brainrather than in the nerve endings). Aspirin and the other non-steroidalanti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases, leading to adecrease in prostaglandin production. This reduces pain and alsoinflammation (in contrast to paracetamol and the opioids). Paracetamolhas few side effects and is regarded as safe, although intake above therecommended dose can lead to liver damage, which can be severe andlife-threatening, and occasionally kidney damage. NSAIDs predispose topeptic ulcers, renal failure, allergic reactions, and occasionallyhearing loss, and can increase the risk of hemorrhage by affectingplatelet function. The use of aspirin in children under 16 sufferingfrom viral illness has been linked to Reye's syndrome, a rare but severeliver disorder. Morphine, the archetypal opioid, and various othersubstances (e.g., codeine, oxycodone, hydrocodone, dihydromorphine,pethidine) all exert a similar influence on the cerebral opioid receptorsystem. Dosing of all opioids may be limited by opioid toxicity(confusion, respiratory depression, myoclonic jerks and pinpoint pupils)and seizures (tramadol), but there is no dose ceiling in patients whoaccumulate tolerance.

The analgesic choice is also determined by the type of pain: forneuropathic pain, traditional analgesics are less effective, and thereis often benefit from classes of drugs that are not normally consideredanalgesics, such as tricyclic antidepressants and anticonvulsants.Tricyclic antidepressants, especially amitriptyline, have been shown toimprove treatment of pain in what appears to be a central manner.Nefopam is used in Europe for pain relief with concurrent opioids. Theexact mechanism of carbamazepine, gabapentin and pregabalin is similarlyunclear, but these anticonvulsants are used to treat neuropathic painwith differing degrees of success. Anticonvulsants are most commonlyused for neuropathic pain as their mechanism of action tends to inhibitpain sensation.

However, certain combination analgesic products can result insignificant adverse events, including accidental overdoses, most oftendue to confusion which arises from the multiple (and often non-acting)components of these combinations (Murnion, Combination analgesics inadults. Australian Prescriber (33): 113-5. See Worldwide Website:australianprescriber.com/magazine/33/4/113/5).

Inadequate treatment of pain is widespread throughout surgical wards,intensive care units, accident and emergency departments, in generalpractice, in the management of all forms of chronic pain includingcancer pain, and in end of life care. This neglect is extended to allages, from neonates to the frail elderly. Improved treatments of painare highly requested by patients, in particular when consideringneuropathic, inflammatory and/or chronic pain for which treatmentremains incomplete whatever the selected known analgesic molecule.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide novel and efficientcompositions and methods for treating pain. In particular, the presentinvention proposes new compositions and methods for preventing ortreating pain by modulating neuronal excitability.

An object of the invention more specifically relates to a TAFA4 protein,or an agonist thereof, for use as an active ingredient for treating orpreventing pain in a subject.

The invention also relates to a method of preventing or treating pain ina subject, the method comprising administering to a subject in needthereof an effective amount of a TAFA4 protein or an agonist thereof,either alone or in combination with one or more additional activecompound(s) efficient against pain.

Another object of the invention relates to a kit comprising i) a TAFA4protein or an agonist thereof or a composition comprising such a proteinor agonist, and ii) at least one additional distinct active compoundefficient against pain.

In a particular embodiment, the TAFA4 protein comprises the amino acidsequence of SEQ ID NO: 1 or 2, or a sequence having at least 90%identity to SEQ ID NO: 1 or 2. The agonist preferably comprises apeptide fragment of a TAFA4 protein that modulates excitability ofnociceptors or interneurons, preferably C-fiber nociceptors (preferablyC-LTMRs) or spinal cord interneurons (preferably spinal cord lamina IIiinterneurons). Typically, the agonist is a peptide comprising a fragmentof at least 10 consecutive amino acid residues of SEQ ID NO: 1 or 2,preferably of at least 20, 25, 27 or 30 consecutive amino acid residues;more preferably the agonist comprises the amino acid sequence of SEQ IDNO: 3 or 4.

In this regard, a further object of the invention relates to apolypeptide or peptide agonist of a TAFA4 protein. Preferably, thepolypeptide agonist comprises a fragment of a TAFA4 protein andmodulates excitability of nociceptors or interneurons, preferablyC-fiber nociceptors (preferably C-LTMRs) or spinal cord interneurons(preferably spinal cord lamina IIi interneurons).

The invention is suitable for preventing or treating any pain. Inparticular, it may be used to treat or prevent neuropathic pain,inflammatory pain, nociceptor-mediated pain, acute pain, subacute pain,chronic pain, somatic pain, visceral pain, allodynia, hyperalgesia, orpain associated with a nerve injury. The invention is particularlysuited for treating inflammatory and/or neuropathic pain.

The TAFA4 protein or agonist may be administered or applied by anyroute, such as a topical, oral, anal, intramuscular, intravenous,intraperitoneal, cutaneous, subcutaneous, dermical, transdermic, orintrathecal route.

A further object of the invention relates to a composition comprising aTAFA4 protein or an agonist thereof as described herein and, preferably,a pharmaceutically acceptable carrier.

The TAFA4 compounds of the invention may be used either alone or infurther combination with one or several additional active compound(s) ortreatment(s). The compounds for use in the present invention may beformulated or administered simultaneously, separately or sequentially.

A further object of this invention relates to a transgenic rodent havinga defective TAFA4 gene, more preferably a targeted inactivated TAFA4gene. Such rodents preferably exhibit an enhanced mechanical andchemical hypersensitivity and enhanced neuronal hyperexcitability.

The invention may be used for treating pain in any mammalian subject,particularly in human subjects.

LEGENDS TO THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication, withcolor drawing(s), will be provided by the Office upon request andpayment of the necessary fee.

FIG. 1: TAFA4 specifically marks C-LTMRs.

(A) Percentage of TAFA4-positive neurons in L4 (n=3; 7.5%±1.3%) and T12(n=3; 19.2%±0.5%) DRG of wild-type adult mice.

(B) Schematic representation of DRG Tafa4 expression data. The sizes ofthe circles in the diagram are roughly proportional to the sizes of thecell populations depicted by the different molecular markers.

(C-H) In situ hybridization for Tafa4 probe in adult mouse lumbar (C-F)and thoracic (G, H) DRG sections followed by immunostaining or in situhybridization for TrkA (C), cRet (D), MrgprD (E), D34(fluorescein-conjugated G. simplicifolia IB4-lectin) (F), TH (G) andEGFP (H). (Scale bars=100 μm.)

FIG. 2: GFP⁺ neurons displayed many properties of C-unmyelinatednociceptors.

(A) Dot plots of the cell membrane capacitance (Cm) and input resistance(R_(input)) of TAFA4 ^(G1+) (GFP⁺) neurons.

(B, C) Recordings of isolated Nav1.8-, T-type Ca²⁺-, IK_(A)- andh-currents (B) and corresponding frequency histograms (C) inTAFA4^(GFP/+) neurons. Number of neurons tested is indicated.

(D) Representative action potentials and firing responses inTAFA4^(GFP1+) neurons evoked by short 2 ms-depolarizing steps (leftpanel) or long duration depolarizing and hyperpolarizing steps (rightpanel). Note Ih-mediated sag on membrane hyperpolarization and thedelayed rebound potential triggered by T-type Ca²⁺ current. The dottedline indicates 0 mV level.

(E) Percentage of GFP⁺-(TAFA4^(GFP/+)) and GFP⁻ neurons that respond toa variety of sensory stimuli (as indicated). Right panel: representativeexamples of calcium signals evoked in TAFA4^(GP+) neurons in response tobath-applied hypotonic solution (200 mOsmol/l) and AITC (100 μM).

(F) Representative traces of MA currents elicited by a standardmechanical stimulus of 8 μm in 4 different TAFA4^(GFP/+) neurons. Thevelocity of the mechanical probe was 800 μm/s during the forward motionof the mechanical stimulus. Holding potential: −100 mV. Right panel:frequency distribution of rapidly adapting (RA), slowly adapting (SA),ultra-slowly adapting (uSA) and mixed MA currents. Data collected over33 TAFA4^(GFP/+) neurons and stimulated with a standard mechanicalstimulus of 8 μm.

(G) Velocity-related firing property of a TAFA4^(GFP/+) neuron. Notethat firing was enhanced as mechanical stimuli were applied with slowrates of onset (n=7).

FIG. 3: Tissue injury-induced hypersensitivity is increased inTAFA4-null mice.

(A) Mechanical threshold of TAFA4-null mice (n=12) and WT littermates(n=11) using dynamic Von Frey apparatus, before and after CFA injection.

(B-E) Time course of mechanical sensitivity of TAFA4-null mice (n=12)and WT littermates (n=7) before (Day0) and following carrageenaninjection using 4 different filaments of increasing calibers (0.07, 0.4,0.6 and 1.4 g). At D+7, the score is before and 15 minutes afterintrathecal injection of 2 μg of human recombinant TAFA4.

(F-H) Time course of mechanical sensitivity following sciatic nerveconstriction (CCI) of TAFA4-null mice (n=12) and WT littermates (n=13)using 3 different filaments of increasing calibers (0.07, 0.6 and 1.4 g,n=15). Measures were determined before (Day0) and every 5 days after theCCI. At D+30, the score is before and 15 minutes after intrathecalinjection of 2 μg of human recombinant TAFA4 (TAFA4-null mice (n=5), WT(n=7)).

(I, J) Time course and total time (in 2 phases: first 0-10 min andsecond 10-60 min) spent in flinching, biting and licking behaviorfollowing 2% formalin injection (WT n=11 and TAFA4-null mice n=12).

(K) Intrathecal injection of 2 μg of human recombinant TAFA4 restoresformalin-evoked hypersensitivity to WT levels in TAFA4-null mice(Vehicle n=8, hTAFA4 n=8). Data shown are mean ±SEM. *p<0.05, **p<0.01,***p<0.001 one-way ANOVA followed by unpaired t-test.

FIG. 4: Lamina IIi neuron excitability in TAFA4-null mice.

(A1) Representative recordings showing the responses of neurons from WT(top) or TAFA4-null (bottom) spinal slices to a 2 s depolarizing (+25)or hyperpolarizing (−25 pA) current pulse.

(A2) Quantification of the average number of AP elicited by currentpulses of increasing intensities (5 to 50 pA). (ANCOVA, p<0.01).

(A3) Average firing rate at different times of the discharge elicited bya 2 s depolarizing current pulse (+25pA) in lamina Ili neurons of WT andTAFA4-null mice.

(A4) Average number of rebound action potentials following a 2 shyperpolarizing current pulse (−50 and −25 pA). (t-test, p<0.05.)

(B1) Representative current responses from WT and TAFA4-null neurons toa back and forth voltage ramp from −40 to −100 mV. Each trace representsthe average of 5 consecutive responses. (B2) Quantification of the peakof the outward current measured at the end of the rising voltage ramp inlamina Iii neurons of WT and TAFA4-null animals (t-test, p<0.05).

(C1) Response of a TAFA4-null lamina IIi neuron to a back and forthvoltage ramp in WT conditions, and in the presence of 20nM recombinantTAFA4, TEA (2.5mM), and 4AP (1mM).

(C2) Quantification of the outward current measured at the end of therising edge of the voltage ramp in TAFA4-Lamina IIi neurons. Notice theincrease in outward current following TAFA4 application (p<0.05), andthe blockade of this current by 4AP.

(D1, D2) Representative traces and quantification of the outward currentin lamina Iii neurons of TAFA4-null animals following the bathsuperfusion of TAFAS and TAFA2 (20nM each).

(E1) Occurrence of low threshold outward current in WT and followingrecombinant TAFA4 superfusion.

(E2) Examples of lamina GAD65/67 negative (left) and positive (right)neurons. Images are single confocal planes. White arrows indicate thelabeling of GAD-positive soma.

FIG. 5: Generation and presentation of TAFA4 GFP mice.

(A) Schematic representation of TAFA4GFP BAC-based strategy used totrigger homologous recombination in Tafa4 locus for the generation ofTAFA4 knock-in mice.

(B-C) In situ hybridization with Tafa4 probe on adult thoracic DRGsections from WT (B) and TAFA4-null mice (C) (n=5).

(D) Total number of neuronal counts in WT DRG (n=3, 6209+/−385 in T12and 7616 +/−173 in L4) and TAFA4-null mice (n=3, 5933+/−324 in T12 and7805+/−439 in L4).

(E, F) Percentage of Ret, TH, TrkA, TrkB, TrkC and TAFA4-positiveneurons in T12 (B) and L4 (C) DRG of WT and TAFA4-null adult mice (n=3).

(G, H) Immunostaining of GFP on transversal section of newbornTAFA4^(GFP/+) (G) and on whole mount adult DRG (H).

(I, I′) Immunostaining of GFP and PKCγ with IB4 staining on lumbarspinal cord sections from adult TAFA4^(GFP/+) mice (I) and TAFA4-nullmice (I′) (n>3).

(J, J′) Immunostaining of GFP and S100 on back skin sections ofTAFA4^(GFP/+) (J) or TAFA4-null adult mice (J′) (n>3).

Scale bars=100 μm. P>0.1 one-way ANOVA followed by unpaired t-test.Error bars represent SEM.

FIG. 6: TAFA4-null mice behave normally in terms of motor activity,anxiety, itch, and acute and injury-induced thermal hypersensitivity.

TAFA4-null mice display unaltered phenotype compared to WT littermatemice in anxiety with open field test (n=14 WT, n=17 TAFA4-null) (A), inmotor coordination with rotaroad test (n=22 WT, n=21 TAFA4-null) (B), inhot plate test (n=18 WT, n=17 TAFA4-null) (C), in acute thermalsensitivity with gradient test (n=15 WT, n=17 TAFA4-null) (D), inCFA-induced thermal hyperalgesia (n=12 WT, n=14 TAFA4-null) (E) and initch test after injection of 100 μg of the pruritogenic agent 48/80 (F).P>0.1 one-way ANOVA followed by unpaired t test. Error bars representSEM.

FIG. 7: Passive properties and low threshold cationic currents inTAFA4-null lamina IIi neurons.

(A) Average values of membrane potential (A1), cell input resistance(A2), cell capacitance

(A3) and rheobase (A4) in lamina IIi neurons of WT and TAFA4-nullanimals.

(B1) Representative recordings showing the Ih sag evoked by a −25 pAhyperpolarizing current pulse in WT and TAFA4-null animals.

(B2) Quantification of the average Ih sag evoked by −50 and −25 pAhyperpolarizing current pulses. (B3) Relation between peak andsteady-state potentials during a hyperpolarizing pulse (range −5 pA-50pA) in lamina Iii neurons of WT and TAFA4-null animals.

(C1) Representative isolated T-type-like current responses of WT andTAFA4-null lamina IIi neurons evoked by depolarizing voltage steps ofincreasing amplitude (25 to 60 mV) from a holding potential of −100 mV.

(C2) Quantification of T-type-like current densities revealedsignificant differences between WT and TAFA4-null mice (t-test; p<0.05).

(C3, C4) T-type currents measured in TAFA4-null neurons before and afterbath application of human recombinant TAFA4.

FIG. 8: Analgesic effect of intrathecal TAFA4 protein in vivo in acarrageenan model of pain.

FIG. 9: Analgesic effect of intrathecal TAFA4 protein in vivo in an SNImodel of neuropathic pain.

FIG. 10: Response of contra-lateral paws following intrathecal injectionof TAFA4.

FIG. 11: Analgesic effect of subcutaneous TAFA4 protein in vivo in acarrageenan model of pain.

FIG. 12: Analgesic effect of subcutaneous TAFA4 protein in vivo in anSNI model of neuropathic pain.

FIG. 13: Analgesic effect of subcutaneous TAFA4 protein in vivo in anSNI model of neuropathic pain 7 (FIG. 13A), 14 (FIG. 13B), and 21 (FIG.13C) days post-surgery.

FIG. 14: Weight monitoring.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new therapeutic approaches for treatingpain. More particularly, the invention provides a new solution toefficiently manage pain, in particular neuropathic pain and inflammatorypain. This solution involves a modulation of sensory sensitivity and/orneuronal excitability by using a TAFA4 compound.

TAFA4 is a small secreted protein belonging to the family of TAFAchemokine-like proteins discovered very recently (Tang et al., 2004).TAFA4 is synthesized as a 140 amino acid precursor that contains a 35amino acid signal sequence and a 105 amino acid mature chain. HumanTAFA4 has 90% amino acid identity with mouse TAFA4. Real-time PCRanalysis indicates that TAFA4 mRNA expression is restricted to thecentral nervous system (CNS), with the highest level in the thalamus.

WO 2006/013462 relates to several gene sequences and uses thereof.However, the proposed uses of these genes, particularly NsG28, areessentially speculative and based merely on expression profiles. In thisregard, while the reference mentions pain, there is no rationale forsaid use and no experimental data to support any such activity which, atleast for some of these genes, turns out to be erroneous. Furthermore,the reference does not disclose the effect or use of isolated proteins,so that a skilled artisan would not consider this document as providingany technical teaching.

Before the present invention, the biological functions of TAFA familymembers remained to be determined.

The inventors have now surprisingly demonstrated, for the first time,that TAFA4 protein is involved in the control of pain. Moreparticularly, the invention shows that TAFA4 is specifically expressedin a particular subset of dorsal root ganglia (DRG) neurons calledC-LTMRs (C-fibers low threshold mechanoreceptors). The invention furthershows that TAFA4-null mice present sustained mechanical and chemicalhypersensitivity following tissue injury, both of which can be reversedby administration of a human recombinant TAFA4 protein.

The inventors have also demonstrated that TAFA4-null mice presentsignificant hyperexcitability of inner lamina II spinal cord neurons.Without being bound by theory, the inventors believe that, in responseto painful stimuli, under pathological conditions, elevated neuronalactivity in C-LTMRs enhances the secretion of TAFA4 protein thatmodulates the excitability of specific interneurons of the spinal cordthrough the activation of a low threshold current.

Interestingly, the inventors have also demonstrated, in the experimentalpart, that TAFA4 protein can specifically target mechanically andchemically induced nociceptive signals, without targetingtemperature-induced signals. This is a considerable advantage incomparison to known pain-treating products which are less specific sincethey also target thermo-induced nociception.

TAFA4 compounds and compositions of the invention are capable ofactivating a new analgesic pathway by modulatingC-LTMR-nociceptor-mediated excitability of spinal cord interneurons(preferably lamina Iii interneurons), for example, via modulation of theactivity of receptors present on said interneurons (such as potassiumion channels, calcium ion channels or low density lipoprotein receptors,e.g., LRP1).

The invention will be best understood by reference to the followingdefinitions:

DEFINITIONS

Within the context of the present invention, the term “TAFA4 compound”designates a TAFA4 protein or a TAFA4 agonist as defined below.

As used herein, the term “TAFA4 protein” designates a protein belongingto the family of TAFA chemokine-like proteins, preferably comprising theamino acid sequence of SEQ ID NO: 1 (which corresponds to the humanTAFA4 amino acid sequence) or SEQ ID NO: 2 (which corresponds to themouse TAFA4 amino acid sequence), and any natural variant thereof (e.g.,variants present in other animal species, or variants as a result ofpolymorphism or splicing). Within the context of the present invention,the term “TAFA4 protein” also includes any protein comprising a sequencehaving at least 90% sequence identity to the sequence shown in SEQ IDNO: 1 or 2, preferably at least 95% sequence identity or more.Typically, a TAFA4 protein is able to modulate nociceptor sensitivityand/or neuronal excitability, as defined below.

Within the context of the present invention, the term “TAFA4 gene”designates a gene or nucleic acid that codes for a TAFA4 protein. Inparticular, a “TAFA4 gene” includes any nucleic acid encoding a proteincomprising SEQ ID NO: 1 or 2, or a natural variant of such a protein.

The term “TAFA4 agonist”, within the context of the present invention,encompasses any substance having, mediating or regulating TAFA4 activity(for example, a peptide, a polypeptide, a recombinant protein, aconjugate, a chemokine, an antigen, a natural or artificial ligand, ahomolog, a nucleic acid, DNA, RNA, an aptamer, etc., or a combinationthereof). In particular, TAFA4 agonists modulate the activity of areceptor involved in TAFA4 activity, for example by binding such areceptor, and thus modulating neuronal excitability. The term “agonist”encompasses both full and partial agonists. Typically, “TAFA4 agonist”designates any compound that can modulate sensitivity of sensory neurons(preferably C-LTMRs) and/or excitability of interneurons (preferablyspinal cord Iii interneurons), in particular by modulation of theactivity of receptors present on said neurons, as described in thepresent application.

“TAFA4 agonist” encompasses any peptide fragment of a TAFA4 protein thatmodulates excitability of nociceptors or interneurons, preferably ofC-fiber nociceptors (preferably C-LTMRs) or spinal cord interneurons(preferably spinal cord lamina IIi interneurons). Typically, a TAFA4agonist is a peptide comprising a fragment of less than 60 amino acidresidues of SEQ ID NO: 1 or 2. Preferably, a TAFA4 agonist comprises atleast 10 consecutive amino acid residues of SEQ ID NO: 1 or 2,preferably of at least 20, 15, 25, 27, 28 or 30 consecutive amino acidresidues. In the most preferable embodiment, such a fragment is afragment comprising at least 10, 15, 20, 25, 27, 28 or 30 consecutiveamino acid residues of the N-terminal part of a “TAFA4 protein” asdefined above. In another embodiment, a TAFA4 agonist comprises at least10, 15, 20, 25, 27, 28 or 30 consecutive amino acid residues of theC-terminal part of a “TAFA4 protein” as defined above. Specific examplesof a TAFA4 agonist are: (i) a peptide comprising the amino acid sequenceof SEQ ID NO: 3 (which corresponds to 25 amino acid residues of theN-terminal part of the human TAFA protein of SEQ ID NO: 1); and (ii) apeptide comprising the amino acid sequence of SEQ ID NO: 4 (whichcorresponds to 27 amino acid residues of the C-terminal part of thehuman TAFA protein of SEQ ID NO: 1). A TAFA4 agonist of the invention isable to modulate nociceptor sensitivity and neuronal excitability, as inthe present application. The term “sequence identity” as applied tonucleic acid or protein sequences refers to the quantification (usuallyas a percentage) of nucleotide or amino acid residue matches between atleast two sequences aligned using a standardized algorithm such asSmith-Waterman alignment (Smith and Waterman (1981) J Mol Biol147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res22:4673-4680), or BLAST2 (Altschul et al. (1997) Nucleic Acids Res25:3389-3402). BLAST2 may be used in a standardized and reproducible wayto insert gaps in one of the sequences in order to optimize alignmentand to achieve a more meaningful comparison between them.

The term “pain”, within the context of the present invention, refers toany pain or sensitivity associated with tissue damage. Preferably, theterm “pain” as used herein is understood as an abnormal sensitivity,i.e., typically as a hypersensitivity which is mediated by nociceptors(in particular by C-LTMRs). The term “pain” includes any pain selectedfrom a nociceptor-mediated pain (also called herein a nociceptive pain),a neuropathic pain, an inflammatory pain, a pathological pain, an acutepain, a subacute pain, a chronic pain, a mechanical pain, a chemicalpain, a somatic pain, a visceral pain, a deep somatic pain, asuperficial somatic pain, a somatoform pain, allodynia, hyperalgesia, ora pain associated with a nerve injury. “Nociceptive” pain or“nociceptor-mediated” pain occurs in response to the activation of aspecific subset of peripheral sensory neurons (nociceptors) by intenseor noxious stimuli. Nociceptive pain according to the invention includesmechanical pain (crushing, tearing, etc.) and chemical pain (iodine in acut, chili powder in the eyes). Examples of nociceptive pain include butare not limited to traumatic or surgical pain, labor pain, sprains, bonefractures, burns, bumps, bruises, injections, dental procedures, skinbiopsies, and obstructions. Nociceptive pain includes visceral pain,deep somatic pain and superficial somatic pain. Visceral pain isdiffuse, difficult to locate and often referred to a distant, usuallysuperficial, structure. It may be accompanied by nausea and vomiting andmay be described as sickening, deep, squeezing, and dull. Deep somaticpain is initiated by stimulation of nociceptors in ligaments, tendons,bones, blood vessels, fasciae and muscles, and is dull, aching, poorlylocalized pain. Examples of deep somatic pain include sprains and brokenbones. Superficial pain is initiated by activation of nociceptors in theskin or other superficial tissue, and is sharp, well-defined and clearlylocated. Examples of injuries that produce superficial somatic paininclude minor wounds and minor (first degree) burns. Inflammatory painis pain that occurs in the presence of tissue damage or inflammationincluding post-operative pain, post-traumatic pain, arthritic(rheumatoid or osteoarthritis) pain and pain associated with damage tojoints, muscles, and tendons as in axial low back pain. Inflammation isresponsible for the sensitization of peripheral sensory neurons, leadingto spontaneous pain and invalidating pain hypersensitivity. Acute orchronic pathological tissue inflammation strongly impacts painperception by sensitizing peripheral sensory neurons, giving rise tolocal and incapacitating pain hypersensitivity. Inflammatory mediatorsare known to enhance nociceptive primary afferent fiber excitability, inpart by modifying the expression and/or function of ion channels presentin nerve endings. Neuropathic pain is a common type of chronic,non-malignant pain, which is the result of an injury or malfunction inthe peripheral or central nervous system. Neuropathic pain may havedifferent etiologies, and may occur, for example, due to trauma,surgery, herniation of an intervertebral disk, spinal cord injury,diabetes, infection with herpes zoster (shingles), HIV/AIDS, late-stagecancer, amputation (including mastectomy), carpal tunnel syndrome,chronic alcohol use, exposure to radiation, and as an unintended sideeffect of neurotoxic treatment agents, such as certain anti-HIV andchemotherapeutic drugs. It is often characterized by chronic allodynia(defined as pain resulting from a stimulus that does not ordinarilyelicit a painful response, such as light touch) and hyperalgesia(defined as an increased sensitivity to a normally painful stimulus),and may persist for months or years beyond the apparent healing of anydamaged tissues. Pain may also occur in patients with cancer, which maybe due to multiple causes: inflammation, compression, invasion, ormetastatic spread into bone or other tissues. Pain also includesmigraine and headaches associated with the activation of sensory fibersinnervating the meninges of the brain. Preferably, TAFA4 compounds ofthe invention are used for preventing or treating a neuropathic and/orinflammatory pain.

“Threshold” of pain, within the context of the present invention,designates the minimum stimulus necessary to produce pain. Inparticular, the pain perception threshold is the point at which thestimulus begins to hurt, and the pain tolerance threshold is reachedwhen the subject acts to stop the pain. For example, pain thresholds aremeasured by gradually increasing the intensity of a stimulus such aselectric current or heat applied to the body.

The term “nociceptors”, within the context of the present invention,designates all possible sensory neurons that mediate nociceptiveinformation relative to pain. Nociceptors innervate cutaneous tissues.The term “nociceptors” includes, without limitation, mechanoreceptors,mechano-nociceptors, multimodal nociceptors, chemoreceptors and/orpruriceptors that detect and transduce a variety of noxious stimuli,including chemical, thermal or mechanical stimuli or combinations ofthese stimuli. A specific example of nociceptors according to theinvention correspond to the low-threshold mechano-receptor (C-LTMR),specifically responding to mechanical and chemical stimuli.

Nociceptors may be modulated by TAFA4 compounds of the invention, or mayuse such compounds to further modulate the excitability of specificinterneurons of the spinal cord, for example, through the activation ofa low threshold current.

The term “interneurons”, within the context of the invention, designatesrelay neurons which transmit information between other neurons.Preferably, interneurons are neurons that relay nociceptive informationfrom sensory neurons to spinal cord projection neurons. The preferredinterneurons according to the invention are spinal cord lamina IIiinterneurons. The interneurons are neurochemically diverse; for example,they can be excitatory interneurons (using glutamate) and/or inhibitoryinterneurons (using GABA or glycine).

Typically, interneurons according to the invention are interneurons thatare directly and/or indirectly modulated by TAFA4 compounds as describedin the present application. The interneurons express varioushistochemical markers and various types of receptors, includingendocytic receptors, metabotropic receptors, inotropic receptors, growthfactor receptors, as well as other signaling molecules, and interneuronexcitability is preferably modulated via modulation of the activity ofsuch receptors.

The term “receptor”, within the context of the present invention,includes any receptor selected from metabotropic receptors, endocyticreceptors (for example, LRP1 receptors) and ionotropic receptors such asligand-gated ion channels and voltage-gated ion channels (for example,potassium channels, calcium channels and sodium channels), or acombination thereof, the activity of which can be modulated by TAFA4compounds of the invention. Endocytic receptors include receptors thatmediate the internalization of a variety of extracellular macromoleculesand macromolecular complexes, including lipoproteins, proteinases,proteinase-inhibitor complexes and extracellular matrix proteins. Aspecific example of such endocytic receptor of the invention is thelow-density lipoprotein receptor-related protein 1 (LRP1 receptor).Endocytic receptors include receptors that are also involved inligand-mediated signal transduction. Ionotropic receptors include ionchannels, channel-linked receptors and ligand-gated ion channels(LGICs). Voltage-gated ion channels (such as potassium channels, sodiumchannels, and calcium channels) are channels playing a fundamental rolein neuronal excitability which are directly responsible for initiationand propagation of action potentials, and implicated in differentchronic pain disorders. LGICs include a group of transmembrane ionchannels that are opened or closed in response to the binding of achemical messenger (i.e., a ligand), such as a neurotransmitter. Thebinding site of endogenous ligands on LGIC protein complexes arenormally located on a different portion of the protein (an allostericbinding site) compared to where the ion conduction pore is located. Thedirect link between ligand binding and opening or closing of the ionchannel, which is characteristic of ligand-gated ion channels, iscontrasted with the indirect function of metabotropic receptors, whichuse second messengers. LGICs are also different from voltage-gated ionchannels (which open and close depending on membrane potential) andstretch-activated ion channels (which open and close depending onmechanical deformation of the cell membrane). Metabotropic receptorscomprise a large protein family of transmembrane receptors that sensemolecules outside the cell and activate inside signal transductionpathways and, ultimately, cellular responses. Metabotropic receptorsinclude G protein-coupled receptors (GPCRs), also known asseven-transmembrane domain receptors, heptahelical receptors, serpentinereceptors, and G protein-linked receptors (GPLR).

The term “modulation” or “modulation of neuronal excitability”, withinthe context of the present invention, designates a change in sensitivityand/or excitability of neurons involved in transmission of pain signalsby using TAFA4 compounds of the invention. The term “modulation”includes a “decrease” of neuronal excitability and/or an “increase” ofneuronal excitability, depending on the type of interneurons theactivity of which is modulated. Neurons which can be modulated by TAFA4are sensory neurons and/or interneurons. Neurons may be modulated byTAFA4 directly or indirectly, electrically or chemically, via receptorsor via ion channels, or by any combination of the above modulatorymodes. An example of nociceptor modulation is a nociceptor modulationcomprising a control of the threshold of somatic sensation in responseto mechanical or chemical stimuli. An example of interneuron modulationis a modulation of interneuron excitability comprising a modulation ofthe activity of receptors present on spinal cord interneurons.

Within the context of the present invention, the term “treatment” or“treating” pain in a subject designates delaying, stabilizing, curing,healing, alleviating, relieving, altering, ameliorating, improving,remedying or affecting any form of pain in a subject as describedherein, or any disease or condition associated with pain (in particularany neuropathic condition associated with neuropathic pain), or anysymptom of such a disease or condition, after the application oradministration of a suitable TAFA4 compound or composition according tothe invention. The term “treatment” or “treating” also refers to anyindicator of success in the treatment of pain (which may be associatedwith any injury, pathology or condition), including any objective orsubjective parameter such as abatement, remission, slowing progressionor severity, stabilization, diminishing of symptoms of pain, or makingit more tolerable to the subject. The term “treating” pain also includesincreasing pain tolerance and/or decreasing perceived pain. Inparticular embodiments, the methods, compounds and composition of theinvention are for increasing pain tolerance and/or for decreasingperceived pain. As used herein, the term “pain tolerance” refers to theamount of pain that a subject can perceive and withstand before breakingdown emotionally and/or physically. Pain tolerance is distinct from painthreshold (the minimum stimulus necessary to produce pain). As usedherein, “increasing pain tolerance” generally refers to a situationwhere a subject can develop a greater pain tolerance (that is, lessperceived pain) when compared to a previous state, for instance,following administration of suitable TAFA4 compounds or compositions toa subject.

Within the context of this invention, “preventing” or “prevention” inrelation to pain in a subject refers to at least the reduction oflikelihood of the risk of (or susceptibility to) acquiring any kind ofpain by a subject, after the application or administration of a suitableTAFA4 compound or composition according to the invention. For example,“preventing” includes causing at least one of the clinical symptoms ofpain not to develop in a subject that may be exposed to or predisposedto, but does not yet experience or display symptoms of pain.

Active Ingredient

An object of the present invention is a TAFA4 compound for use as anactive ingredient for preventing or treating pain in a subject, inparticular neuropathic pain, inflammatory pain, acute pain, sub-acutepain, chronic pain, allodynia, hyperalgesia, partially treated pain,chemically induced pain, and mechanically induced pain, as well asrefractory pains, while preferably advantageously avoiding deleteriousside effects. In a preferred embodiment, the TAFA4 compound is toefficiently manage neuropathic pain or inflammatory pain. The compoundsaccording to the invention may also be used to prevent or treat chronicpain in subjects suffering from pathologies such as cancer, burns, etc.,for which generally analgesics (such as morphine) may be administeredfor a long period, optionally in delayed form. The compounds accordingto the invention may also be used together with reduced daily doses ofmorphine in order to improve the clinical picture of patients (bylimiting side effects of morphinomimetics, such as intestinal disorders,for example).

In a preferred embodiment, the compound of the invention is a TAFA4protein comprising the sequence of SEQ ID NO: 1 or a sequence having atleast 90% identity to SEQ ID NO: 1, or a TAFA4 agonist, preferablycomprising a fragment of SEQ ID NO: 1, more preferably at least a 30aafragment thereof as indicated in the sequence of SEQ ID NO: 2, thatmodulates the activity of at least one receptor present on spinal cordlamina IIi interneurons (in particular, the low density protein LRP1receptor or a potassium channel, a calcium channel, or anotherphysiologically relevant receptor). In a particular embodiment, theTAFA4 compounds of the invention advantageously modulate the activity ofan additional receptor (distinct from the first receptor).

Subject

In the context of the present invention, the patient or subject is ananimal, preferably a vertebrate, typically a mammal. In a preferredembodiment, the mammal is a human being of any age or sex. The mammalmay further be an animal, in particular a domestic or breeding animal,in particular a horse, a dog, a cat, etc. In a particular embodiment,the subject suffers from a neuropathic pain or an inflammatory pain, inparticular a chronic inflammatory pain. In another particularembodiment, the subject is afflicted with any disease or conditionassociated with pain, initiated in any manner.

Compositions

The invention also relates to a pharmaceutical composition comprising aTAFA4 compound as herein described as an active ingredient, andpreferably a pharmaceutically acceptable carrier.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention (active ingredient) and a medium generally accepted in theart for the delivery of biologically active compounds to a subject inneed thereof. Such carriers include all pharmaceutically acceptablecarriers, diluents, media or supports therefore. This carrier can beselected for example from methyl-beta-cyclodextrin, a polymer of acrylicacid (such as carbopol), a mixture of polyethylene glycol andpolypropylene glycol, monoethanolamine and hydroxymethylcellulose.Conventional pharmaceutical practice may be employed to provide suitableformulations or compositions to subjects, for example in unit dosageform.

This composition is typically a local analgesic/anti-hyperalgesiccomposition.

The compounds and compositions of the invention are adapted for use forpreventing, alleviating or treating pain in a subject as describedabove.

The composition of the invention can further comprise at least oneadditional active compound. This compound can be advantageously selectedfrom a SAID, NSAID or opioid drug.

In another embodiment, a compound or composition of the invention canalso be administered, for example, along with an agent intended to treata coincident condition (e.g., an antitumor agent).

The compounds for use in the present invention may be administeredsimultaneously, separately or sequentially.

Methods of Production of Compounds of the Invention

The present invention also concerns methods of production of TAFAcompounds.

TAFA4 compounds of the invention (e.g., protein or peptide agonists) canbe produced by any conventionally known protein expression method andpurification method, for example: (i) a method for synthesizingpeptides; (ii) a method for purifying and isolating them from the livingbody or cultured cells; or (iii) a method for producing them with theuse of genetic recombination techniques, and the like (for example, thestandard techniques described for example in Molecular Cloning(Sambrook, J., Fritsch, E. F., Maniatis, T., Cold Spring HarborLaboratory Press) (1989) and Current Protocols in Molecular Biology(Ausubel, F. M., John Wiley and Sons, Inc. (1989)). Preferred proteinsor agonists for use in the invention are therefore isolated or purified.As commonly used, “isolated” indicates for instance that the protein oragonist is at least separated from some components of its natural orproduction environment such as a cell culture medium or living organism.More preferably, the proteins or agonists are used as isolated or purematerial with a purity level above 50%, above 60%, above 70%, above 80%,above 90%, or even more preferably above 95%. The isolated or purifiedprotein or agonist may then be combined or mixed with additionalingredients such as excipients or further active agents, as described insubsequent sections.

Treatment/Protocol/Regimen

Also taught herein is a method for the prevention or treatment of painin a subject. An aim of the method is modulating neuronal excitabilityusing TAFA4 compounds or compositions as defined above. A particularmethod for preventing, alleviating or treating a nociceptor-mediatedpain (in particular a C-LTMR-mediated pain) in a subject in need thereofcomprises administering to the subject an effective amount of a TAFA4compound or composition as herein described.

A further particular method for preventing, alleviating or treating painin a subject in need thereof comprises a step of administering to saidsubject a TAFA4 compound or composition, as herein described, in atherapeutically effective amount, possibly in combination with at leastone additional active compound such as any one of the moleculesmentioned in the background part, for example aspirin, ibuprofen,paracetamol, opioid, etc.

Preferably, the treatment method refers to treating neuropathic pain orneuropathy, comprising any improvement in the symptoms of such aneuropathy or any retardation or reduction of outward signs, for examplea reduction of their frequency, of the trouble or discomfort, of pain,or even total disappearance of the neuropathy. In a particularembodiment, the TAFA4 compounds or compositions of the invention areuseful in preventing neuropathic pain or preventing neuropathy beforedevelopment of the first signs of the disease in order to protect asubject from such a neuropathic pain or neuropathy to which the subjectis, or may be, exposed.

The duration, dosages and frequency of administering compounds orcompositions of the invention for such a treatment may be also adaptedaccording to different forms of pain (i.e., acute or chronic neuropathicpain). The treatment may be used alone or in combination with otheractive ingredients, either simultaneously, separately or sequentially.

The compounds or compositions according to the invention may beadministered in various ways or routes. The compounds or compositions ofthe invention may be administered by intramuscular, intravenous,intraperitoneal, cutaneous, subcutaneous, dermal, transdermal,intrathecal, ocular (for example corneal) or rectal ways, or by atopical administration on an inflammation site, and preferably byintramuscular or intravenous injection.

A typical regimen comprises a single or repeated administration of aneffective amount of a TAFA4 compound over a period of one or severaldays, up to one year, and including between one week and about sixmonths, or it may be chronic. It is understood that the dosage of apharmaceutical compound or composition of the invention administered invivo will be dependent upon the age, health, sex, and weight of therecipient (subject), kind of concurrent treatment, if any, frequency oftreatment, and the nature of the pharmaceutical effect desired. Theranges of effectives doses provided herein are not intended to belimiting and represent preferred dose ranges. However, the mostpreferred dosage will be tailored to the individual subject, as isunderstood and determinable by one skilled in the relevant arts (see,e.g., Berkowet et al., eds., The Merck Manual, 16^(th) edition, Merckand Co., Rahway, N.J., 1992; Goodmanetna., eds., Goodman and Cilman'sThe pharmacological Basis of Therapeutics, 10^(th) edition, PergamonPress, Inc., Elmsford, N.Y., (2001)).

The total dose required for each treatment can be administered bymultiple doses or in a single dose, preferably as soon as the earlysymptoms of pain appear, or preventively, for example before or duringsurgery when needed. The pharmaceutical compound can be administeredalone or in conjunction with at least one other pharmaceutical directedto the pathology, or directed to other symptoms of the pathology.Effective amounts of a compound or composition according to theinvention are from about 1 μg to 100 mg/kg body weight, preferablyadministered at intervals of 4-24 hours for a period of several days,weeks, months, or up to 1 year, and/or any range or value therein, suchas 0.001-0.01, 0.01-0.1, 0.05-100, 0.05-10, 0.05-5, 0.05-1, 0.1-100,0.1-1.0, 0.1-5, 1.0-10, 5-10, 10-20, 20-50, and 50-100 mg/kg, forexample between 0.05 and 100 mg/kg, preferably between 0.05 and 5 mg/kg,for example 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4 or 5 mg/kg, atintervals of 1-4, 4-10, 10-16, or 16-24 hours, for a period of 1-14,14-28, or 30-44 days, or 1-24 weeks, or any range or value therein. Atypical administration schedule comprises from 1 μg to 100 mg/kg/day.

The recipients of administration of compounds and/or compositions of theinvention can be any subjects as herein defined, preferably humans.

Formulations/Concentrations

The compounds or compositions according to the invention may beadministered in various forms. Thus, they may be formulated in the formof ointments, gels, pastes, liquid solutions, suspensions, tablets,gelatin capsules, capsules, suppositories (in particular for painassociated with a gastrointestinal syndrome), powders, nasal drops, oraerosols, preferably in the form of an ointment. The compounds of theinvention are typically administered in the form of ointments, gels,oils, tablets, suppositories, powders, gelatin capsules, capsules, etc.,optionally by means of dosage forms or devices that ensure prolongedand/or delayed release. For this type of formulation, an agent such ascellulose, carbonate or starch is advantageously used. For injections,the compounds are generally packaged in the form of liquid suspensions,which may be injected via syringes or perfusions, for example. In thisrespect, the compounds are generally dissolved in saline, physiological,isotonic or buffered solutions, etc., compatible with pharmaceutical useand known to the person skilled in the art. Thus, the compositions maycontain one or more agents or excipients selected from dispersants,solubilizers, stabilizers, preservatives, etc. Agents or excipients thatcan be used in liquid and/or injectable formulations are notablymethylcellulose, hydroxymethylcellulose, carboxymethylcellulose,polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, etc.It is understood that the flow rate and/or dose administered may beadjusted by the person skilled in the art according to the patient, thepain observed, the area to be treated, the active compound(s) concerned,the mode of administration, etc.

For topical applications, it is preferred to expose the subject to betreated to an effective amount of a pharmaceutical compound orcomposition according to the invention to target areas, e.g., skinsurfaces, mucous membranes, and the like, which are adjacent to theperipheral neurons to be treated.

Typically, the compounds are administered at doses that may vary betweenabout 50 _(j—)Ig to about 5 mg/kg of body weight of a compound of theinvention, depending upon the previously mentioned criteria, whether theuse is prophylactic or therapeutic and the nature of the topical vehicleemployed. A preferred administration is an intramuscular, intravenous orintraperitoneal injection. Furthermore, administration by injection maycomprise several (2, 3 or 4) administrations per day, if need be. Inaddition, for chronic treatments, delayed or prolonged systems may beadvantageous, ensuring the subject effective and long-lasting paintreatment.

The invention also relates to a kit comprising (i) a TAFA4 compound orcomposition, as previously described, (ii) at least one additionaldistinct active compound efficient against pain, and optionally (iii)written instructions for using the kit.

According to a specific embodiment, the invention also relates to a kitthat is suitable for treatment by the methods herein described. Thesekits comprise (i) a TAFA4 compound or composition, as previouslydescribed, typically in the dosages herein indicated, and (ii) a secondcomposition containing an analgesic compound, preferably an opiatecompound, in dosages generally lowered when compared to thoseclassically prescribed, for simultaneous, separate or sequentialadministration, in effective amounts according to the invention.

The figures and examples illustrate the invention without limiting itsscope.

EXAMPLES I. Experimental Procedures

All animals (mice) were maintained in standard housing conditions (22°C., 40% humidity, 12 h light cycles, and free access to food and water).Special effort was made to minimize the number as well as the stress andsuffering of mice used in the below experiments. All protocols are inagreement with European Union recommendations for animalexperimentation.

I.1. Generation of Tafa4-GFP KI Mice with Targeted Inactivated TAFA4Ggene

To generate tafa4-GFP KI mice we used the Bacterial ArtificialChromosome (BAC)-based homologous recombination in embryonic stem cells.The final targeting vector was constructed on the basis of a 209 kbgenomic clone of the mouse tafa4 locus in pBAC (RP23-427L8), obtainedfrom a 129SVJ “BACPAC” Resources Center (BPRC) library (FIG. 2A). Thebacterial recombination in the RP23-427L8 BAC vector was engineeredthanks to an intermediate targeting construct that has been assembledusing the plasmid vectors pL452 and pCS2/venus sv40. 109 bp of the tafa4gene exonl was replaced by the “YFP (Venus, hereafter GFP)-polyAloxP-EM7-PGK-Neo-loxP” cassette. The arms of homology were isolated as271 pb and 265 pb PCR products using Taq phusion polymerase (Finnzymes).After homologous recombination of the BAC in bacteria, the finaltargeting construct was linearized using the Ascl site and transfectedinto 129/SV-derived embryonic stem cells CK35. Homologous recombinantclones were identified by Southern blot using probes located at the 3′end of the construct, and by a neomycin probe. Two targeted clones wereinjected into C57B16/J derived blastocysts at the Immunology Centertransgenic facility. Resulting chimeras were mated to C57B16/J femalesto produce germ line transmission of the recombinant allele.

The following oligonucleotides were used for genotyping PCRs:

GFP 436: (SEQ ID NO: 28) GAAGAAGTCGTGCTGCTTCATGTG, 1585: (SEQ ID NO: 29)CTGTGGAGGAAATGGTTTCAACT, and 1587: (SEQ ID NO: 30)CTGCAAAGAGAAGCCAAAGCTAC.

Heterozygous males and females were mated to generate the populationdescribed in behavioral tests of the manuscript. In order to increasethe visualization of GFP for cellular and molecular experiments, we alsogenerated a TAFA4 GFP-NEO-line. The neo cassette was removed by crossingTAFA4^(GFP/+) mice to a cre-deleter mouse line. The absence of neocassette was confirmed by PCR. Except for behavioral analyses andwhole-cell patch-clamp recording from spinal cord slices with attacheddorsal root, Cre recombined TAFA4 GFP mice were used for allexperiments.

I.2. Tissue Sections and In Situ Hybridization/Immunofluorescence

To obtain adult tissues, mice were deeply anesthetized with a mix ofketamine/xylazine and then transcardially perfused with an ice-coldsolution of paraformaldehyde 4% in PBS (PAF). After dissection, theywere postfixed for at least 24 h in the same fixative at 4° C. PO werecollected in ice-cold PBS 1X, gently washed, and fixed for 24 h in 4%PAF. For skin immunofluorescence, trunk skin was excised fromanesthetized mice and fixed directly in 15% (v/v) acid picric-2%formaldehyde for 24 h at 4° C. Tissues were then transferred into a 30%(w/v) sucrose solution for cryoprotection before being frozen and storedat -80° C. Samples were sectioned (12 to 40 p.m) using a standardcryostat (Leica). In situ hybridization and immunofluorescence wereperformed following standard protocols (Mocrich et al., 2004). RNAprobes (Tafa4, TH, Vglut3, TrkB, MrgprD, SCG10) were synthesized usinggene-specific PCR primers and cDNA templates from embryonic or adultmouse DRG. More particularly, in situ hybridization was performed usinga combination of digoxigenin and/or fluorescein/biotin labeled probes.Probes were hybridized overnight at 55° C., and the slides incubatedwith the horseradish peroxidase anti-digoxigenin/fluorescein/biotinantibody (Roche). Final detection was achieved using afluorescein/cy3/cy5 TSA Plus kit (PerkinElmer). For double-fluorescentin situ experiments, the first antibody was inactivated using H₂O₂treatment.

The following oligonucleotides were used for the nested PCRs for probesynthesis:

tafa4-F1: (SEQ ID NO: 5) TGCTCAGAAGTTCATAGCCAAA, tafa4-R1:(SEQ ID NO: 6) TAAAGGAACATTTGCAAGCTCA, tafa4-F2: (SEQ ID NO: 7)ATATGTGCAGTGTGG, tafa4-R2 + T7: (SEQ ID NO: 8)TAATACGACTCACTATAGGGCAGCCAAGTTCAAAC, TH-F1:  (SEQ ID NO: 9)AAGCCAAAATCCACCACTTAGA, TH-R1:  (SEQ ID NO: 10) CCGTGGAGAGTTTTTCAATTTC,TH-R2 + T7: (SEQ ID NO: 11) TAATACGACTCACTATAGGGAGAGATGCAAGTCCAATGTCCT,Vglut3-F1: (SEQ ID NO: 12) TAGCTCAGTTTCCAGGAATGGT, Vglut3-R1:(SEQ ID NO: 13) GGAGATCTAACAACATCTGATAACAC, Vglut3-F2: (SEQ ID NO: 14)CCCCCTAGAGTATCAGGAATTT, Vglut3-R2 + T7: (SEQ ID NO: 15)TAATACGACTCACTATAGGGTGGGAAGTTTTAAAAATCTATGATTAG, TrkB-F1:(SEQ ID NO: 16) CTGAGAGGGCCAGTCACTTC, TrkB-R1: (SEQ ID NO: 17)CATGGCAGGTCAACAAGCTA, TrkB-F2: (SEQ ID NO: 18) CAGTGGGTCTCAGCACAGAA,TrkB-R2 + T7: (SEQ ID NO: 19) TAATACGACTCACTATAGGGCTAGGACCAGGATGGCTCTG,MrgprD-F1: (SEQ ID NO: 20) GGGCATCAACTGGTTCTTACTC, MrgprD-R1:(SEQ ID NO: 21) AGGGATTGTCTTGACTGTCG, MrgprD-F2: (SEQ ID NO: 22)AACGGGATGTGAGGCTACTTTA, MrgprD-R2 + T7: (SEQ ID NO: 23)TAATACGACTCACTATAGGGATTTATGCCTTGACTTCCCTGA, SCG10-F1: (SEQ ID NO: 24)GCAATGGCCTACAAGGAAAA, SCG10-R1: (SEQ ID NO: 25) GGCAGGAAGCAGATTACGAG,SCG10-F2: (SEQ ID NO: 26) AGCAGTTGGCAGAGAAGAGG, and SCG10R2 + T7:(SEQ ID NO: 27) TAATACGACTCACTATAGGGGGCAGGAAGCAGATTACGAG.

For immunofluorescence, primary antibodies were diluted in PBS—10%donkey or goat serum (Sigma)—3% bovine albumin (Sigma)—0.4% Triton X-100and incubated overnight at 4° C. Primary antibody concentrations andreferences are: rabbit anti-TrkA 1:1000 (Interchim), goat anti-TrkC1:1000 (R&D Systems), goat anti-Ret 1:500 (R&D Systems), rabbitanti-CGRP 1:2000 (Chemicon), chicken anti-green fluorescent protein(GFP) 1:1000 (Ayes Labs), rabbit anti-PKCy 1:1000 (Santa CruzBiotechnology), anti-S100 1:400 (Dako), and goat anti-parvalbumin 1:1000(Swant). Corresponding donkey or goat anti-rabbit, anti-chick, andanti-goat Alexa 488, 555, or 647 (Invitrogen or Molecular Probesantibodies) were used for secondary detection. Isolectin D34 conjugatesto Alexa FluorR 568 dye were used at 1:100 (Invitrogen).

I.3. Cell Counts and Statistical Analysis.

12 μm serial sections of thoracic (T12) and lumbar (L4) DRG weredistributed on six and eight slides respectively and subjected todifferent markers including the pan-neuronal marker SCG10. Thisapproach, in addition to providing the total number of neurons, allowedus to represent all counts as a percentage of SCG10⁺ neurons. For eachgenotype, two to four DRG were counted in at least three independentmice. Statistical significance was set to p <0.05 and assessed usingone-way ANOVA followed by unpaired t-test.

I.4. Electrophysiological Recording and Calcium Imaging

Whole-cell patch-clamp recording of cultures of DRG neurons and fromspinal cord slices with attached dorsal root as well as calcium imagingprotocols are described below.

-   -   Cultures of DRG Neurons for Patch Clamp Recording

7 to 14 week old heterozygous or TAFA4-null male mice were anesthetizedwith halothane and sacrificed by severing of the carotid arteries inaccordance with the Guide for the Care and Use of Laboratory Animals.Dissociation and cultures of DRG neurons were realized from lumbar DRGsexcised and freed from their connective tissue sheaths as previouslydescribed (Hao and Delmas, 2010, 2011). They were incubated in enzymesolution containing 2 mg/ml of collagenase IA for 45 min at 37° C. andtriturated in Hanks' medium (GIBCO BRL). The resulting suspension wasplated in Nunclon dishes coated with 10 ng/ml laminin (Sigma). Culturemedium was Dulbecco's modified Eagle's medium (DMEM) supplemented with10% heat-inactivated FCS, 100 U/ml penicillin-streptomycin, 2 mM1-glutamine, 25 ng/ml nerve growth factor (NGF7S, Sigma-Aldrich,France), and 2 ng/ml glial-derived neurotrophic factor (GDNF,Invitrogen, France) (all from GIBCO BRL). Neurons were maintained in ahumidified atmosphere (5% CO₂, 37° C.) for 12 h before recording.

-   -   Whole-Cell Patch-Clamp Recording

Patch clamp recordings were performed using borosilicate electrodeshaving resistances ranging from 2 to 3 MQ. Recording of Nav1.8 and ICaTused a CsCl-based pipette solution consisting of (mM): 125 CsCl, 10HEPES, 5 NaCl, 0.4 NaGTP, 4 MgATP, 1 MgCl₂, 4.8 CaCl₂ and 10 EGTA(adjusted to pH 7.3 with CsOH). IKA, h-current and MA currents wererecorded using a KCl-based pipette solution containing (mM): 134 KCl, 10HEPES, 4 MgATP, 0.4 NaGTP, 1 MgCl₂, 4.8 CaCL₂ and 10 EGTA (pH 7.3). Thesame KC1-based pipette solution was used for current-clamp recording.The standard external solution consisted of (mM): 132 NaCl, 1 KC1, 1MgCl₂, 2.5 CaCl₂, 10 HEPES, 10 D-glucose and TTX (500 nM, AscentScientific) (adjusted to pH 7.3 with NaOH, 300 mOsm/1). Neurons wereperfused with bath solution at a flow rate of 2-3 ml/min.

-   -   Mechanical Stimulation

Mechanical stimulation using piezoelectrically driven mechanical probeshas been detailed elsewhere (Hao and Delmas, 2010). Briefly, afire-polished glass micropipette cemented to a piezo-electric actuator(Step Driver PZ-100; Burleigh) was used as a mechanical probe andpositioned at an angle of 45° from horizontal. Downward movement of theprobe toward the cell was driven by pClamp program (Molecular Devices).The probe had a velocity of 800 μm/s (unless otherwise noted) during theramp segment of the command for forward motion, and the stimulus wasapplied for durations ranging from 200 ms to several seconds. Unlessotherwise noted, voltage-clamped MA currents were recorded at a holdingpotential of −100 mV.

The time constants of MS current decay were fitted to exponentials usingthe Chebyshev nonlinear least square fitting procedure (Hao and Delmas,2010). Current traces were fitted with either monoexponential orbi-exponential functions. Bi-exponential functions were as follows:I(t)=A1·exp(−t/τ1)+A2·exp(−t/τ2)+Ao, where τ1 and τ2 represent the rapidand slow exponential components, Al and A2 represent the amplitude ofeach respective component, and Ao represents the baseline current. Fitswith greater than two exponential components did not significantlyenhance description of the current decay, as judged by residualanalysis. Cells were classified as expressing a particular MS cationcurrent if the main component (>80%) of the current evoked at −100 mVdeclined monoexponentially. MS currents not meeting this requirementwere classified as mixed. Based on current decay time constants, threetypes of MS currents could be distinguished: rapidly adapting currents(IR, 3-6 ms), slowly adapting currents (IS, 200-300 ms) and ultra-slowlyadapting currents (IuS, ≥1000 ms).

-   -   Data Acquisition and Analysis

Voltage and current recordings were made using an Axopatch 200Bamplifier (Molecular Devices), filtered at 1 kHz, and sampled at 40-100μs. Voltage errors were minimized using 75-85% series resistancecompensation. Cell capacitance was estimated from the time constant ofthe decay phase of a current transient elicited by a 10 mVhyperpolarizing step. All experiments were done at room temperature.PRISM 4.0 (GraphPad) software was used to perform data analysis. Resultsare presented as mean ±SEM and n represents the number of neuronsexamined. Statistical analysis used Student's t-test and P<0.01 wasconsidered statistically significant.

-   -   Whole-Cell Patch-Clamp Recording from Spinal Cord Slices with        Attached Dorsal Root

Transverse spinal cord slices with attached dorsal roots from juvenile(P21 to P34) TAFA4-null and WT mice were prepared for whole-cellrecording following the protocol described in Mourot et al. (2012). Micewere deeply anesthetized with isoflurane before being quickly beheaded.A piece of tissue containing the spinal column and surrounding muscleswas quickly removed and immersed in ice-cold oxygenated low-calciumartificial cerebrospinal fluid (ACSF) (in mM: NaCl 101; KCl 3.8; MgCl₂18.7; MgSO₄ 1.3; KH₂PO₄ 1.2; HEPES 10; CaCl₂ 1; glucose 1). Afterlaminectomy, the spinal cord was gently removed and its lumbar part wasplaced into a small 3% agarose block. Spinal slices (300 μm thick) werecut using a Leica VTS1000 vibratome, and transferred into warm (31° C.)ACSF (in mM: NaCl 130.5; KCl 2.4; CaCl₂ 2.4; NaHCO₃ 19.5; MgSO₄ 1.3;KH₂PO₄ 1.2; HEPES 1.25; glucose 10; pH 7.4) equilibrated with 95% O₂-5%CO₂ for at least one hour before starting patch clamp recordings. Spinalslices were placed in a recording chamber bathed with warmed (31° C.)ACSF. Electrophysiological measurements were performed under the controlof an Olympus BX51 microscope using a 2B multiclamp (Molecular Devices).Patch pipettes (7-11 S2) were filled with appropriate pipette solution(in mM: K-gluconate 120; KCl 20; CaCl₂ 0.1; MgCl₂ 1.3; EGTA 1; HEPES 10;GTP 0.1; cAMP 0.2; Leupeptin 0.1; Na2ATP 3; D-Manitol 77; pH 7.3). Forthe measurement of T-type calcium currents, the patch pipette had thefollowing concentrations (in mM: Cs methanesulfonate 120; CsC1 20; CaCl₂0.1; MgCl₂ 1.3; EGTA 1; HEPES 10; GTP 0.1; cAMP 0.2; leupeptin 0.1;Na2ATP 3; D-mannitol 77; pH 7.3), and TTX (0.5 μM), CNQX (5 μM), DL-APV(10 μM), strychnine (10 μM), bicuculline (5 ₁.tM) and TEA (2.5 mM) wereadded to the ACSF in order to block sodium voltage activated andsynaptic currents. A glass suction electrode connected to a Master 8(A.M.P. Instruments Ltd.) stimulator was used to stimulate dorsal roots.Typically, high duration (500 μs), high intensity (350 μA) stimulationswere used to recruit most primary afferent fibers in the recorded slice.Liquid junction potentials (calculated value −16.5 mV) are not correctedfor.

-   -   Molecular Identification of Spinal Lamina IIi Recorded        Interneurons

To determine the neurotransmitter phenotype of recorded lamina IIneurons, biocytin was added 0.5% to the pipette recording solution. Atthe end of the recordings, the patch pipette was carefully removed topreserve as much as possible the integrity of the recorded neurons.Spinal slices were then fixed overnight at 4° C. in 4% PFA and kept at−4° C. in 0.5% PFA for later revelation of biocytin and GAD. The sliceswere rinsed 3 times in PBST and incubated in primary antibody(anti-GAD6567, Sigma G5163, 1/2000 in PBST 0.5%BSA) for 48 hours at 4°C. The slices were rinsed 3 times in PBST and incubated overnight in amix of secondary antibody (goat anti-rabbit Alexa 568, Molecular ProbesA-11011, 1/500) and streptavidin Alexa 488 (Invitrogen 5-11266, 1/500).The slices were rinsed 3 times in PBST and mounted in Dako fluorescentmounting medium. Acquisitions were performed on a Leica SPE confocalmicroscope using x63 oil immersion objectives.

-   -   Ca2+ Imaging

Lumbar DRG neurons from heterozygote or knockout 7 to 14 week old TAFA4mice were seeded on laminin coated glass-bottom chambers (FluoroDish,WPI) and cultivated for 16-22 hours at 37° C. in B27 supplementedNeurobasal A medium (Invitrogen, France) with 100 ng/ml NGF 7S(Sigma-Aldrich, France), 2 ng/ml GDNF (Invitrogen, France), and 10 ng/mlNT4 (PeproTech, France). Calcium imaging was performed 12-17 hours afterseeding. Prior to recording, neurons were incubated with 5 μM fura-2AMin Tyrode's solution for 1 hour at 37° C. Fluorescence measurements weremade with an inverted microscope (Olympus IX70) equipped with a CoolSNAPHQ camera (Roper Scientific, France). Fura-2 was excited at 340 nm and380 nm and ratios of emitted fluorescence at 510 nm were acquiredsimultaneously with bath temperature using MetaFluor software (UniversalImaging). Temperature was controlled with a gravity driven perfusion(1-2 ml/min) cooled with a Peltier device mounted in series with aresistive heater (CellMicroControls). Perfusion was first cooled at 12°C. than heated at 37° C. before application onto the chamber.Temperature was monitored with a thermistor probe located near theperfusion outlet always at the same place. Rapid cooling from 37° C. toless than 15° C., achieved by switching off the heating, took typicallyless than 40 sec. Pharmacological agonists of several transient receptorion channels (100 μM menthol, 100 μM allyl isothiocyanate (AITC), 0.5 μMcapsaicin, 10 μM pregnenolone sulfate) were prepared into the Tyrode'ssolution and applied sequentially to the neurons for a few seconds at37° C. For iso- and hypotonic stimulations, the extracellular solutionswere prepared keeping constant the concentration of NaCl and varying thelevel of mannitol to control the osmolarity. The isotonic solution(300mOsm) contained (in mM): 87 NaCl, 100 mannitol, 3 KCl, 1 MgCL₂, 2.5CaCl₂, 10 HEPES, and 10 glucose, and the hypotonic solution (20 mOsm)contained (in mM): 87 NaCl, 51 mannitol, 3 KCl, 1 MgCl₂, 2.5 CaCl₂, 10HEPES, and 10 glucose. Data were analysed offline using MetaFluor,Excel, and GraphPad Prism.

II. Behavioral Assays

All behaviour analyses (Open field, Rotarod, Hot plate, Cold plate,Thermal gradient, Two-temperature choice, Thermal nociceptive threshold(Hargreaves' test), Itch test, Von Frey, Dynamic Von Frey and Formalintest) were conducted on littermate males 8-12 weeks old. Detaileddescription of all these tests is provided below. Complete Freund'sadjuvant (CFA) and carrageenan hindpaw injection, intrathecal injectionof recombinant TAFA4 and chronic constriction of the sciatic nerve (CCI)are also described below. Student's t-test was used for all statisticalcalculations.

More particularly, all behavioral assays were conducted on littermates8-12 weeks of age of mixed C57BL6/129SV genetic background. Animals wereacclimated for 20 minutes to their testing environment prior to allexperiments, which were done at room temperature (˜22° C.).Experimenters were blind to the genotype of the mice during testing.Student's t-test was used for all statistical calculations. All errorbars represent standard error of the mean (SEM). General behavioral(locomotor and learning activity) was measured using a rotarod apparatus(LSI Letica Scientific Instruments). Gradient, thermal plates, openfield, Hargreaves' and Von Frey apparatus were from Bioseb.

II.1. General Behavioral Assays

-   -   II.1.A. Open Field Test

The open field test is commonly used to assess locomotor, exploratoryand anxiety-like behavior. It consists of an empty and bright squarearena (40×40×35 cm), surrounded by walls to prevent the animal fromescaping. The animals were individually placed in the center of thearena and their behavior recorded with a video camera over a 5-minuteperiod. Anxiety-related behavior is measured by the degree to which therodent avoids the center area (20×20 cm), analysed by Bioseb trackingsoftware.

-   -   II.1.B. Rotarod Test

A rotarod apparatus (LSI, Letica Scientific Instruments) was used toexplore coordinated locomotor, balance and learning function in mice.Mice were placed on a rod that slowly accelerated from 4 rpm to 44 rpmconstant speeds of rotation over 5 min, and the latency to fall offduring this period was recorded. The test was done 4 consecutive days.Each day, the animals were tested three times separated by at least a 5min resting period. Response to temperature choice test and response totemperature gradient assay were performed as described in Moqrich et al.(2005) but using a Bioseb apparatus.

II.2. Thermal Sensitivity Tests

-   -   II.2.A. Hot Plate

To assess heat sensitivity, mice were confined individually to a metalsurface maintained at 48°, 50° or 52° C. by a Plexiglass cylinder 20 cmhigh, and the latency to nociceptive responses (licking, shaking orjumping of hind paws) measured. To prevent tissue damage, mice wereremoved from the plate immediately after a nociceptive response or aftera maximum of 90 s, 60 s and 45 s respectively. Each mouse was tested twotimes with a latency of 5 min between each test; the withdrawal timecorresponds to the mean of the two measures. A latency of at least lhbetween each tested temperature was respected.

-   -   II.2.B. Cold Plate

To test cold sensitivity, mice were placed individually into aPlexiglass chamber maintained at 22°, 10° or 4° C. The rearing time ofthe mice during the first minute is measured. Each mouse is exposedthree times to each temperature with a minimum five-minute restingperiod between trials and one hour separating periods betweentemperatures.

-   -   II.2.C. Thermal Gradient Test

This test has been described previously (Moqrich et al., 2005). Briefly,mice were individually tracked for 90 min in four separate arenas of thethermal gradient apparatus

(Bioseb). A controlled and stable temperature gradient of 14° C. to53.5° C. was maintained using two Peltier heating/cooling devicespositioned at each end of the aluminum floor. Each arena was virtuallydivided into 15 zones of equal size (8 cm) with a distinct and stabletemperature. The tracking was performed using a video camera controlledby the software provided by the manufacturer.

-   -   II.2.D. Two-Temperature Choice Tests

Two mice were placed simultaneously in each lane of the two-temperaturechoice apparatus (Bioseb). Mice were tracked for 10 min using the Biosebsoftware. During the first day, both plates were kept at 20° C. for 10min. Days after this acclimatizing period, 2 plates were individuallywarmed or cooled to different temperature (42° C. to 16° C.) and kept atthe appropriate temperature for the 10 min test. A 1 h time lapse wasrespected between 2 different tests.

-   -   II.2.E. Thermal Nociceptive Threshold (Hargreaves' Test)

To assess hind paw heat sensitivity, Hargreaves' test was conductedusing a plantar test device (Bioseb). Mice were placed individually intoPlexiglass chambers on an elevated glass platform and allowed toacclimatize for at least 30 minutes before testing. A mobile radiantheat source of constant intensity was then applied to the glabroussurface of the paw through the glass plate and the latency to pawwithdrawal measured. Paw withdrawal latency is reported as the mean ofthree measurements for both hind paws with at least a 5 min pausebetween measurements. IR source was adjusted to 20% and a cut-off of 20s was applied to avoid tissue damage.

II.3. Mechanical Sensitivity Testing

-   -   II.3.A. Dynamic Von Frey

To assess hind paw mechanical sensitivity, the dynamic Von Frey test wasconducted using a Bioseb apparatus. Von Frey filament is applied with anincreasing strength up to 7 g for 20 s. Injected and non-injected hindpaws are pinched three times with at least 5 min of latency between andthe average of withdrawal (g or second) is calculated.

-   -   II.3.B. Von Frey Filament Test

For the chronic constriction model, we used the Von Frey hair filamentsof three different bending forces (0.07, 0.6 and 1.4 g). For thecarrageenan model, mechanical allodynia and hyperalgesia were assessedusing the Von Frey hair filaments of four different bending forces(0.07, 0.4, 0.6 and 1.4 g). For details, see the “Unilateral peripheralmononeuropathy” and “Carrageenan inj ection” paragraphs.

II.4. Chemical Sensitivity Testing

-   -   II4.A. Formalin Test

Formalin solution was prepared at 2% in PBS 1X from a formalin stock(Fisher Scientific) (note that formalin stock corresponds to a 37%formaldehyde solution). Mice were housed individually in Plexiglasschambers and allowed to habituate to the testing environment for 30minutes. Following subcutaneous injection of 10 ₁1.1 of formalin in theleft hind paw, the animals were immediately placed individually inobservation chambers and then monitored for pain behavior (shaking,licking and biting of the injected paw) for 60 min. The pain behaviorcumulative time of the injected paw was counted at 5-minute intervals.Time spent exhibiting these pain behaviors was recorded for the firstphase (0-10 min) and the second phase (10-60 min).

-   -   II.4.B. Itch Test Using Pruritogenic Agent 48/80

Pruritogenic agent 48/80 (Sigma-Aldrich, C2313) was prepared at 2 μg/μlin PBS 1X. 100 μg (50 μl) were injected into the mouse's neck. Theitching cumulative time was counted for 40 minutes.

-   -   II.4.C. CFA Injection

10 μl of complete Freund's adjuvant (CFA) was injected into the lefthind paw of anesthetized mice using a Hamilton syringe, in order toproduce inflammation and alterations in nociceptive sensitivity.Injected paws were assessed for signs of acute inflammation, such asedema and redness, 24 hours after injection. The responses to thermaland mechanical stimuli were measured before injection (Dayo), as well asone, three and seven (only for mechanical) days after CFA injection. Theuninjected right hind paws served as a control.

-   -   II.4.D. Carrageenan Injection

20 μl of 1% X-Carrageenan (Sigma-Aldrich, 22049-5G-F) in PBS1X wasinjected into the mouse's left hind paw using a Hamilton syringe.

For the carrageenan model, mechanical allodynia and hyperalgesia wereassessed before and after injection using the Von Frey hair filaments offour different bending forces (0.07, 0.4, 0.6 and 1.4 g). For eachfilament, two times five stimuli were applied with an interval of 3 to 5seconds. The uninjected right hind paws served as a control.

-   -   II.4.E. Unilateral Peripheral Mononeuropathy

For the chronic constriction of the sciatic nerve (CCI) model,unilateral peripheral mononeuropathy was induced in mice anaesthetizedwith Ketamine (40 mg/kg ip) and Xylasine (5 mg/kg ip) with three chromicgut (4_0) ligatures tied loosely (with about 1mm spacing) around thecommon sciatic nerve (Bennett and Xie, 1988).

The nerve was constricted to a barely discernable degree, so thatcirculation through the epineurial vasculature was not interrupted(Descoeur et al., 2011). For the chronic constriction model, mechanicalallodynia and hyperalgesia were assessed before the surgery and everyother 5 days post-surgery using the Von Frey hair filaments of threedifferent bending forces (0.07, 0.6 and 1.4 g). For each filament, twotimes five stimuli were applied with an interval of 3 to 5 seconds.

-   -   II.4.F. Intrathecal Injection of Recombinant TAFA4

Intrathecal (i.t.) injections of TAFA4 (200m/ml, human recombinantTAFA4, R&D Systems) or vehicle (PBS) in a volume of 10 pi were done 15min before the formalin test. Mice were held in one hand by the pelvicgirdle and a 25-gauge needle connected to a 20 μl Hamilton syringe wasinserted into the subarachnoidal space between lumbar vertebrae L5 andL6, until a tail-flick was elicited.

III. RESULTS

III.1.TAFA4 is a Specific Marker of C-LTMRs

Interestingly, the inventors have found that Tafa4 transcripts werehighly enriched in adult DRG and trigeminal neurons. Using in situhybridization, the inventors have demonstrated that Tafa4 transcriptsare expressed in approximately 8% and 19% of total lumbar (L4) andthoracic (T12) adult DRG neurons, respectively (FIG. 1A). Doublefluorescent labeling experiments showed that Tafa4 is completelyexcluded from TrkA⁺ neurons and identifies a subset of Ret⁺ neurons(FIGS. 1C and 1D). TAFA4⁺ neurons do not bind IB4 and are completelydistinct from mrgpror neurons (FIGS. 1E and 1F). In contrast, TAFA4 ispredominantly co-expressed with TH and VGLUT3 (FIG. 1G). UsingVLUT3-EGFP DRG sections (Seal et al., 2009), the inventors have foundthat 92+/−4% of TAFA4⁺ neurons co-express EGFP and 94+/−6% of EGFP⁺neurons co-express TAFA4 (FIG. 1H), identifying TAFA4 as a specificmarker of C-LTMRs. In contrast to TH and VGLUT3, TAFA4 is almostrestricted to DRG and trigeminal neurons with a low expression incentral nervous system neurons, namely in the habenula and in scatteredpopulations of neurons in the nuclei of the brain stem and hypothalamus.

III..2. TAFA4-Expressing Neurons Display Properties ofMechano-Nociceptors

To investigate the role of TAFA4 in C-LTMRs, the inventors havegenerated a knock-in mouse model that allows the genetic labeling ofTAFA4-expressing neurons while eliminating TAFA4 protein in a targetedmanner (i.e., without affecting unknown genes) (FIG. 5A). The inventorsfirst confirmed that TAFA4 transcripts were completely abolished inTAFA4^(GFP/GFP) homozygous mice (herein TAFA4-null mice) (FIGS. 5B and5C). GFP⁺ neurons projected to the innermost layer of lamina IIcentrally and exclusively innervated the hairy part of the skinperipherally (FIGS. 5G-5J).

Using patch-clamp recordings and calcium imaging, the inventors havefound that GFP⁺ neurons displayed many properties of C-unmyelinatednociceptors, including small cell capacitance, high input resistance,short duration action potential devoid of a prominent hump in therepolarizing phase, and a remarkable concomitant expression ofTTX-resistant Nav1.8, low-threshold T-type Ca²⁺ (ICa_(T)), A-type K⁺current (IK_(A)) and hyperpolarization-activated h (I_(h)) currents(FIGS. 2A-2C). ICa_(T)-mediated rebound potentials were also typicallyobserved at repolarization (FIG. 2D). The activation of IK_(A) resultedin a delay in the occurrence of action potentials (APs) or reboundpotentials in response to positive or negative current steps,respectively (FIGS. 2D and 6). The homogeneous presence of thesedifferent currents shapes the cell firing in a unique way, with adepolarizing “sag” response to negative current steps due to I_(h) and a“gap” in AP firing in response to depolarizing current steps. Thesefiring properties can be used as specific criteria to classifyTAFA4-expressing neurons.

GFP⁺ neurons did not respond to many putative nociceptive agents,including capsaicin, menthol, pregnenolone sulfate and 5HT or to rapidcooling (FIG. 2E). In contrast, GFP⁺ neurons displayed differentialresponses to the TRPA1 agonist allyl isthiocyanate (AITC) and tohypo-osmotic solution (FIG. 2E), suggesting some functionalheterogeneity within C-LTMRs.

Classical features of C-LTMRs, including slow conduction velocities,trains of spikes in response to a light mechanical force and slowadaptation to a sustained mechanical stimulus, have been determinedusing ex-vivo skin nerve preparations (Bessou et al., 1971; Li et al.,2011; Seal et al., 2009; Woodbury et al., 2001). Application ofmechanical forces to the cell body of GFP⁺ neurons revealed the presenceof mechanically-activated (MA) cation currents in 95% of neurons tested(FIGS. 2F and 2G). Although rapidly adapting MA currents could beoccasionally encountered (15%), slowly and ultra-slowly adapting MAcurrents were predominant (21.3 and 57.9%, respectively) in GFP⁺ neurons(FIG. 2F). All these currents were cationic and non-selective, withreversal potential ranging from -2 to +4 mV. Consistent with the slowadaptation properties of MA currents, slow velocity ramp stimulus wasable to trigger APs (FIG. 2G), indicating that mechanosensory GFP⁺neurons respond to slow motion stimuli.

In conclusion, all the above expression data, combined with calciumimaging and electrophysiological recordings, demonstrate that TAFA4⁺neurons display physiological properties of C-unmyelinatedmechano-nociceptors.

III..3. TAFA4-Null Mice Develop Severe Injury-Induced Mechanical andChemical Hypersensitivity

To gain insight into the functional role of TAFA4 in C-LTMRs, theinventors have subjected TAFA4-null mice to a large battery ofsomatosensory tests under acute, inflammatory and neuropathic painconditions. TAFA4-null mice appeared normal in terms of body weight,open-field (FIG. 6A) and rotarod (FIG. 6B) profiles, demonstrating thatTAFA4-null mice do not have abnormalities in motor activity or anxiety.The inventors found no difference between WT and TAFA4-null mice in thehot plate (FIG. 6C), thermotaxis gradient assay (FIG. 6D) or Hargreaves'test (FIG. 6E) as well as in the cold plate, the two-temperature choiceand the dynamic cold and hot plate tests. Then the inventors testedTAFA4-null mice for ability to sense mechanical stimuli under acute,inflammatory and neuropathic pain conditions.

In the complete Freund's adjuvant (CFA) model, mechanical sensitivitywas measured using the automated Von Frey apparatus (FIG. 3A). Bothgenotypes exhibited a significant decrease of withdrawal threshold forthe treated paw 24 hours after CFA injection. When tested 3 dayspost-CFA, TAFA4-null mice exhibited a significantly lower withdrawalthreshold compared to WT mice.

Complete recovery for both genotypes was achieved 7 dayspost-inflammation. To further explore the role of TAFA4 in mechanicalsensitivity, the inventors used Von Frey filaments in response tocarrageenan (FIGS. 3B-3E). Consistent with the CFA model, TAFA4-nullmice exhibited prolonged pain hypersensitivity in response to all testedfilaments at 3 and 7 days post treatment. Very interestingly, TAFA4-nullmice displayed enhanced mechanical hypersensitivity as early as 1 and 3hours post-carrageenan treatment with all filaments including the finestcalibers (0.07 and 0.4g), suggesting an important role of TAFA4 intactile allodynia (FIGS. 3B-3E).

Finally, to assess the role of TAFA4 in neuropathic pain, the inventorsused the chronic constriction of the sciatic nerve (CCI) model (FIGS.3F-3H). TAFA4-null mice exhibited a prolonged mechanicalhypersensitivity phenotype for all tested filaments, demonstrating arole for TAFA4 in neuropathic pain.

III4. Human Recombinant TAFA4 Completely Reversed Mechanical andFormalin-Induced Pain Hypersensitivity in TAFA4 Null-Mice

Intrathecal administration of 2 μg of human recombinant TAFA4 seven dayspost-carrageenan or 30 days post-CCI reversed both hypersensitivityphenotypes observed in TAFA4-null mice to WT levels (FIGS. 3B-3H,day7+TAFA4 and day30+TAFA4).

To test whether the enhanced mechanical hypersensitivity in TAFA4-nullmice was modality specific, the inventors carried out the formalin test(FIGS. 31 and 3J). Intraplantar injection of 10 μl of 2% formalintriggered a robust first pain response in both genotypes. TAFA4-nullmice exhibited a dramatically elevated response in the second phase,suggestive of an enhanced central sensitization in these mice.Importantly, formalin-induced hypersensitivity in TAFA4-null mice wasreversed to WT levels after intrathecal administration of TAFA4 fifteenminutes before formalin injection (FIG. 3K).

Taken together, the above results demonstrate that TAFA4 is required tomaintain the normal threshold of injury-induced mechanical and chemicalpain hypersensitivity.

III.5. Lamina IIi Neurons Exhibit Increased Excitability in TAFA4Null-Mice

To further explore the central sensitization phenotype induced by lossof TAFA4, the inventors performed whole-cell recordings of lamina IIineurons in dorsal root-attached spinal cord slices from WT (n=19) andTAFA4-null mice (n=25). However, injection of depolarizing currentpulses of increasing amplitudes (0-50pA) elicited more action potentialsin TAFA4-null neurons than in WT (FIGS. 4A1 and 4A2, ANCOVA, p<0.001).This effect was even more pronounced at the onset of the depolarizingcurrent pulse, as TAFA4-null neurons showed increased dischargefrequency at the beginning of the current pulse, before adapting todischarge rates comparable to those of WT neurons (FIG. 4A3).Furthermore, injection of hyperpolarizing current pulses (−50 or −25 pA)elicited higher rebound AP in TAFA4-null neurons compared to WT (FIGS.4A1 and 4A4, p=0.049 and p=0.001, respectively). Together, these datademonstrate an increased excitability of lamina IIi neurons inTAFA4-null mice.

The differences observed in TAFA4-null mice show a differentialregulation of slowly inactivating low threshold currents. Tocharacterize these currents, the inventors measured the outward currentelicited at −40 mV in lamina IIi neurons using a symmetrical voltageramp protocol (−40 to −120 and back to −40 mV). Whereas in WT neurons anoutward current with slow desensitization could be observed at the endof the rising voltage ramp, this current was almost absent in TAFA4-nullneurons (FIGS. 4B1 and 4B2, p=0.001). As intrathecally administeredrecombinant TAFA4 diminishes the exaggerated pain behavior in injuredTAFA4-null mice, the inventors examined the effects of addingrecombinant human TAFA4 on lamina Iii neurons from TAFA4-null mice. Theinventors found that exogenous application of TAFA4 (20-30 mn, 20 nM)induced the expression of an outward current, similar to that observedin neurons from WT animals in control conditions (i.e., without TAFA4)(FIGS. 4C1 and 4C2, n=19, p<0.001). This current was not affected byexternal TEA (2.5 mM, n=3), but was completely blocked by 4AP (1 mM),thus demonstrating that A-type current pharmacology is involved, i.e.,potassium ionic channels. These effects were specific to TAFA4, asaddition of recombinant TAFAS (n=5, FIGS. 4D1 and 4D2) or TAFA2 (n=6,FIG. 4D2) could not elicit this low threshold outward current fromTAFA4-null neurons.

Following TAFA4 addition, the distribution of outward currentintensities among lamina IIi neurons was best fitted by a mix of twoGaussian curves, revealing the existence of two distinct populations:one-third of the neurons displayed significant outward currents whilethe remaining neurons were weakly or not affected by TAFA4 bathapplication (FIG. 4E1). Phenotypic characterization of TAFA4-responsiveneurons showed that TAFA4 elicited similar outward currents both inGAD-positive and GAD-negative neurons (FIG. 4E2).

The experimental data show that TAFA4 depresses a subset of glutamergicexcitatory (GAD−) and GABAergic inhibitory interneurons (GAD+),preferably by the activation of a low threshold outward currents. Inparticular, as excitatory transmission seems to dominate sensoryprocessing in spinal cord substancia gelatinosa (corresponding to spinalcord lamina II), the net result of such a dual depression ofGABAergiques and glutamergic neurons by TAFA4 would be dominated by adecrease in excitatory transmission, thereby reducing the amount ofnociceptive information transmitted to lamina I projection neurons.Thus, TAFA4 compound according to the invention, reduces nociceptiveinformation by decreasing excitatory transmission in spinal cordinterneurons.

Among low threshold currents, Ih and T-type calcium currents may alsoshape the firing of lamina IIi neurons. To characterize Ih-like currentsin WT and TAFA4-null mice, the inventors quantified thehyperpolarization evoked sag by measuring the difference between peakand steady-state potentials in response to a hyperpolarizing currentpulse (FIG. 7B1). The inventors found that isolated T-type currentsevoked by square potential pulses (see methods) were frequently weakerin WT than in TAFA4-null mice (FIG. 7C1). Statistical analysis revealeda significant increase in T-type current densities in TAFA4-null laminaIIi neurons compared to WT (FIG. 7C2; p=0.001).

Taken together, the above results indicate that TAFA4 modulates theintensity of low-threshold outward currents in lamina IIi neurons,directly or indirectly.

IV. Analgesic Effect of Intrathecal TAFA4 in Animals with NeuropathicPain

IV.1. Neuropathic Pain Model SN1

The SNI model (Spared Nerve Injury, developed by Decosterd and Woolf,2000; Pain, Vol. 87, p 149-158) was used. The SNI model consists of thetransection of tibial branches and of the common peroneal nerve of thesciatic nerve, the sural nerve remaining intact. The latter thendevelops signs of neuropathic pain with substantial mechanicalallodynia. The SNI model has many advantages:

-   -   Neuropathic pain is persistent. This allows the grasp of        habituation phenomena upon repeated injections of TAFA4.    -   The generated pain is robust.    -   The model is very reproducible.

IV.2. Dose-Effect Study

In order to determine the optimal concentration, a first test wasconducted on a “fast” inflammatory pain model (1% carrageenan).

Procedure:

18 eight-week-old male TAFA4-KO mice are used.

Recombinant human TAFA4 (#5099-TA, R&D, batch #PXCO213101) isresuspended in 0.9% NaCl at 3 different concentrations (12.5 μg/mL, 50μg/mL and 200 μg/mL).

Von Frey filament measurement with the up/down method for determiningthe baseline.

Intraplantar injection of 20 μl of carrageenan (1%) in a hind paw.

Measurement of the response threshold 4 h after injection to check forthe occurrence of inflammatory pain.

24 h later, a new measurement is made.

Then blind intrathecal injection of 10 μl of TAFA4 solution at 3different concentrations (n=6 to 12.5 μg/mL; n=5 to 50 μg/mL; n=6 to 200μg/mL) is performed.

Measurement of the response threshold is made 30 minutes afterinjection.

Results:

The results are shown in FIG. 8. Occurrence of mechanical allodynia isobserved 4 hours after injection of carrageenan, and is maintained 24 hlater. Injection of 10 μl of each of the TAFA4 solutions induced astrong increase in the threshold response to Von Frey filaments. Thethree tested concentrations induced a statistically significantreduction in the pain induced by carrageenan (*p<0.05).

These concentrations (12.5, 50 and 200 μg/mL) were used for subsequenttesting of the analgesic effect of TAFA4 by intrathecal injection in theSNI neuropathic pain model.

IV.3. Intrathecal Injection in SNI Animals

Procedure:

The experiments are conducted on eight-week-old WT C57B16 mice. 42 micewere used.

Recombinant human TAFA4 (#5099-TA, R&D, batch #PXCO213101) isresuspended in 0.9% NaCl at 3 different concentrations (12.5 μg/mL, 50μg/mL and 200 μg/mL). A 200 μg/mL

BSA solution is used as a negative control. After having measured thebase threshold of the mice with Von Frey filaments by the up/downmethod, the SNI module is set into place. The mice are anesthetized,ligature of the tibial nerve and the fibular nerve is put into practiceand these two nerves are then severed. The sural nerve left intactdevelops neuropathy quite rapidly. 3 days after surgery, occurrence ofneuropathy is ascertained. A decrease of the response threshold to VonFrey filaments of the ipsilateral paw is thereby observed.

7 days after surgery, the response threshold is again measured. 10 μl ofeach of the 3 TAFA4 solutions (n=10 at 12.5 μg/mL; n=10 at 50 μg/mL; n=9at 200 μg/mL) and of BSA solution (n=10) are then blind-injectedintrathecally.

The response threshold is measured 30 minutes, 2 hours, 4 hours, 6 hoursand 24 hours after injection.

Results:

The results are presented in FIG. 9. After intrathecal injection, asignificant increase of the response threshold for the three TAFA4solutions was observed as soon as 30 minutes after injection. On theother hand, injection of BSA had no effect on the response threshold ofthe mice. After 2 h, the analgesic effect was maintained at its maximumfor the three concentrations. After 4 h, mice having received aninjection of 2 μg TAFA4 still exhibited a high response threshold (**:p<0.01; *: p<0.05). We also monitored the response of contra-lateralpaws following intrathecal injection of TAFA4 or BSA solutions, and nostatistical difference was observed (see FIG. 10).

These results show that intrathecal injection of the three tested TAFA4concentrations caused a substantial comparable analgesic effect onneuropathic pain. The effect of the strongest concentration (200 μg/ml,i.e., 2 μg of TAFA4) lasts longer. Furthermore, TAFA4 did not inhibitthe nerve impulse activity of sensorial neurons as indicated by the lackof a change in response of the contra-lateral paw.

V. Analgesic Effect of Subcutaneous TAFA4 in Animals with NeuropathicPain

This example illustrates the analgesic effect of TAFA4 on a neuropathicpain model following subcutaneous injection.

V.1. Dose-Effect Study

In order to apprehend the doses which may be tested subcutaneously, afirst “fast” test on an inflammatory pain model (1% carrageenan) wasconducted on a restricted number of TAFA4-KO mice.

Procedure:

9 eight-week-old male TAFA4-KO mice are used. Recombinant human TAFA4(#5099-TA, R&D, batch #PXCO213101) is resuspended in 0.9% NaCl at 2different concentrations (10 μg/mL and 30 μg/mL), for a 10 μl injectionper gram. Measurement with Von Frey filaments by the up/down method fordetermining the baseline. Intraplantar injection of 20 μl of carrageenan(1%) into a hind paw. Measurement of the response threshold, 24 h afterinjection, followed by subcutaneous blind injection of TAFA4 solution at2 different concentrations (n=3 at 100 μg/kg; n=3 at 300 μg/kg) orpregabalin solution at 30 mg/kg (n=3), for the experimenter.

Measurement of the response threshold is measured, 30 minutes afterinjection of the compounds, and subsequently 2 h and 4 h.

Results:

The results are shown in FIG. 11. After injection of carrageenan, themice developed mechanical allodynia. Injection of pregabalin caused anincrease in the response threshold. Similarly, injection of TAFA4 alsoinduced a statistically significant increase in the response thresholdat 100 μg/kg and even stronger at 300 μg/kg.

TAFA4 therefore caused an analgesic effect by subcutaneous injection inthe carrageenan model.

V.2. Subcutaneous Injection in SNI Animals

Procedure:

The experiments were conducted on eight-week-old male WT C57B16 mice. 48mice were used. Recombinant human TAFA4 (#5099-TA, R&D, batch#PXCO213101) is resuspended in 0.9% NaCl at 3 different concentrations(3 μg/mL, 10 μg/mL and 30 μg/mL). A 30 μg/mL BSA solution is used as anegative control. After having measured the base threshold of mice withVon Frey filaments by the up/down method, the SNI model is set intoplace. The mice are anesthetized, ligature of the tibial nerve andfibular nerve is put into practice and these two nerves are thensevered. The sural nerve left intact develops neuropathy quite rapidly.The occurrence of neuropathy is ascertained 3 days post-surgery. Adecrease in the response threshold to Von Frey filaments of theipsilateral paw is thereby observed.

7 days after surgery, the response threshold is again measured. 100μl/10 g of each of the TAFA4 solutions (n=11 at 30 μg/kg; n=12 at 100μg/kg; n=11 at 300 μg/kg) and of BSA (n=12) are then blind-injectedsubcutaneously for the experimenter.

The response threshold is measured 1 hour, 2 hours, 4 hours, 6 hours and24 hours after injection.

Results (FIG. 12):

The subcutaneous injection of TAFA4 induced a strong increase in theresponse threshold as soon as 1 hour post-injection. This effect wasmaintained for at least 4 h with the three tested concentrations. As forintrathecal injection, the effect seems to last longer with the higherconcentration.

Subcutaneous injection of TAFA4 therefore induces an analgesic effect onmechanical allodynia induced by the SNI neuropathic pain model. Theconcentration of 300 μg/kg was used for the continuation of the study.

VI. TAFA4 Induces a Sustained Analgesic Effect with No Side Effects

The purpose of these experiments was to further confirm the analgesiceffect of TAFA4 by subcutaneous injection in the SNI model, and to checkthat this effect is maintained and safe by achieving several injectionpoints.

Procedure:

The experiments were conducted on eight-week-old male WT C57B16 mice. 24mice were used. Recombinant human TAFA4 (#5099-TA, R&D, batch#PXCO213101 and #PXCO214011) is resuspended in 30 μg/mL of 0.9% NaCl. A30 μg/mL BSA solution is used as a negative control. After havingmeasured the base threshold of mice with Von Frey filaments by theup/down method, the SNI model is set into place. The mice areanesthetized, ligature of the tibial nerve and fibular nerve is put intopractice and these two nerves are then severed. The sural nerve leftintact develops neuropathy quite rapidly.

7 days after surgery, a decrease of the response threshold to Von Freyfilaments of the ipsilateral paw is observed. 10 μl/g of the TAFA4 (300μg/kg) and BSA solutions are then blind-injected subcutaneously (n=12for each of the BSA and TAFA4 groups). The response threshold ismeasured 1 hour, 2 hours, 4 hours and 6 hours after injection. The sameexperimental procedure is carried out at 7 days, 14 days and 21 dayspost-surgery.

Results (FIG. 13):

A strong increase in the response threshold to mechanical stimulationwas observed following subcutaneous injection of 300 μg/kg of TAFA4 at 7days, 14 days and 21 days post-surgery. About 40-50% of the initialvalue (before surgery) may be reached. In the three cases, the effectremained similar (with no significant difference) as indicated byanalysis of the areas under the curve.

These results therefore confirm the potent analgesic effect of the TAFA4protein or agonist of the invention.

Various organs (liver, spleen, kidneys, heart and lungs) of thesubcutaneously treated animals were removed and frozen for subsequentstudies. The weight of the treated animals was monitored all along theexperiment. No difference was observed in the weight curve (FIG. 14).Furthermore, the various removed organs were weighed with precisionscales, before being set in 4% paraformaldehyde (PFA) overnight at 4°C., and then incubated in sucrose 30% before being cryogenically kept inOCT at −80° C. No difference was observed in all of the tested organs(liver, spleen, kidneys, heart and lungs) between treated and controlanimals.

Our results therefore show the effect of a TAFA4 protein in an SNIneuropathic pain model. Mechanical allodynia (decrease in the responsethreshold) induced by this model may be inhibited by intrathecal orsubcutaneous injection of TAFA4. It is important to note that the dosesused are low (2 μg intrathecally and 6-8 μg subcutaneously). Further,the response threshold of the contra-lateral paws remained unchangedafter injection of TAFA4, thus showing that TAFA4 does not act as anagent blocking nerve impulses.

VII. Conclusions

-   -   The present invention demonstrates, for the first time, that        TAFA4 protein is involved in the control of pain, and shows its        efficiency in the treatment of pain in different pain models.    -   The invention also shows that TAFA4 is specifically expressed in        small-diameter sensory neuron C-LTMRs.    -   The invention further shows that TAFA4 loss-of-function led to        increased injury-induced mechanical and chemical        hypersensitivity and enhanced excitiability of lamina IIi        neurons.    -   The invention also shows that TAFA4⁺ afferents exclusively        innervate hair follicles in the periphery and project to the        innermost layer of lamina II centrally.    -   The invention shows that TAFA4 modulates neuronal excitability        and the threshold of somatic sensation.    -   The invention further shows that TAFA4 protein can specifically        target mechanically and chemically induced nociceptive signals.    -   The invention also shows that TAFA4 compounds and compositions        are capable of activating a new analgesic pathway by modulating        C-LTMR-nociceptor-mediated excitability of spinal cord        interneurons (preferably lamina IIi interneurons), for example,        via modulation of the activity of receptors present on said        interneurons (such as potassium ion channels, calcium ion        channels or low-density lipoprotein receptors, e.g., LRP1).    -   The inventors also propose that TAFA4 may regulate presynaptic        channels in primary afferents which in turn increase synaptic        transmission. Postsynaptically, the “TAFA4ergic” C-LTMR        afferents face a network of lamina Ili excitatory glutamatergic        and inhibitory GABAergic/glycinergic interneurons that are        connected to projection neurons residing in lamina I.    -   The inventors further show that mechanical and formalin-induced        pain hypersensitivity in TAFA4-null mice was reversed to WT        levels after administration of the human recombinant TAFA4.    -   All the experimental data demonstrated by the inventors in the        present application also revealed that C-LTMR-derived TAFA4        modulates the second phase of formalin-evoked pain. In        particular, the data provided herein show that TAFA4-null mice        exhibited exaggerated/enhanced formalin-evoked pain. The        inventors propose that formalin-evoked nocifensive behavior        could be specifically triggered by C-LTMR sensory neurons.    -   Genetic marking of TAFA4-expressing neurons allowed detailed in        vitro study of the physiological properties of C-LTMRs.        Patch-clamp analysis revealed a strikingly homogenous population        of neurons with small capacitance, unique short-duration APs,        the presence of a TTX resistant Nav1.8 current and a remarkable        co-expression of several low threshold currents as well as        slowly and ultra-slowly adapting excitatory mechano-gated        currents.    -   By comparing pain phenotypes in wild-type (m) or TAFA4 null        mice, inventors have established the proof of concept that        disturbing TAFA4 function causes modulation of neuronal        excitability, which contributes to pain signaling. In        particular, the inventors have clearly demonstrated that loss of        TAFA4 function enhances mechanical and chemical hypersensitivity        in a variety of pain models. In conclusion, TAFA4 can be used as        an active ingredient for treating pathological pain signaling        via modulation of neuronal excitability.

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1. A composition comprising a TAFA4 protein or an agonist thereof and apharmaceutically acceptable carrier, wherein said protein comprises theamino acid sequence of SEQ ID NO: 1 or 2 or a sequence having at least90% identity to SEQ ID NO: 1 or 2; and wherein said agonist is a peptidecomprising at least 10 and fewer than 60 consecutive amino acid residuesof SEQ ID NO: 1 or
 2. 2. The composition of claim 1, wherein said TAFA4protein comprises SEQ ID NO: 1 or comprises a sequence having at least90% identity to SEQ ID NO:
 1. 3. The composition of claim 2, whereinsaid TAFA4 protein comprises SEQ ID NO:
 1. 4. The composition of claim1, wherein said composition further comprises at least one additionalactive compound effective against pain.
 5. The composition of claim 1,wherein said composition is a local analgesic and/or anti-hyperalgesiccomposition.
 6. A kit comprising i) a TAFA4 protein or a compositionthereof, wherein said TAFA4 protein comprises the amino acid sequence ofSEQ ID NO: 1 or 2 or a sequence having at least 90% identity to SEQ IDNO: 1 or 2, and ii) at least one additional distinct active compoundefficient against pain.
 7. The kit of claim 6, wherein said TAFA4protein comprises SEQ ID NO: 1 or comprises a sequence having at least90% identity to SEQ ID NO:
 1. 8. The kit of claim 7, wherein said TAFA4protein comprises SEQ ID NO:
 1. 9. A transgenic rodent having a targetedinactivated TAFA4 gene.